LOCUS Exported 5722 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of EYFP fused to the cell penetrating peptide TAT with a GST tag for purification. ACCESSION . VERSION . KEYWORDS SRpHi1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5722) AUTHORS Richardson DS, Gregor C, Winter FR, Urban NT, Sahl SJ, Willig KI, Hell SW TITLE SRpHi ratiometric pH biosensors for super-resolution microscopy. JOURNAL Nat Commun. 2017 Sep 18;8(1):577. doi: 10.1038/s41467-017-00606-4. PUBMED 28924139 REFERENCE 2 (bases 1 to 5722) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5722 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..29 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 37..53 /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)." CDS 76..729 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 687..709 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 736..759 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 808..1524 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(850..871) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1111..1130) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1458..1479 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1590..1612) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1757..1775) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1843..1947 /gene="bla" /label=AmpR promoter CDS 1948..2808 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2166..2185) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2979..3567 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3468..3487 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3721..3738 /label=L4440 /note="L4440 vector, forward primer" promoter 3811..3888 /gene="lacI" /label=lacI promoter CDS 3889..4971 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(3908..3927) /label=LacI-R /note="LacI, reverse primer" promoter 5020..5050 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5058..5074 /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 5082..5098 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5082..5098 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 5094..5621 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR QPSEAVVWLCRS" primer_bind complement(5114..5131) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5114..5130) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5123..5145) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca 61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa 121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc 181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt 241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata 301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt 361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt 421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat 481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg 541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa 601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc 661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat 721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatccta tggccgaaag 781 aagcggcgtc aacgacgtcg agaattcgtg agcaagggcg aggagctgtt caccggggtg 841 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 901 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 961 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc 1021 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1081 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 1141 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1201 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 1261 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 1321 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1381 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc 1441 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 1501 ggcatggacg agctgtacaa gtaactcgag cggccgcatc gtgactgact gacgatctgc 1561 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 1621 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 1681 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat 1741 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 1801 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 1861 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 1921 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 1981 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 2041 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2101 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2161 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc 2221 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2281 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2341 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2401 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2461 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta 2521 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2581 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2641 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2701 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2761 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 2821 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 2881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 2941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 3001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 3061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 3121 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 3181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 3241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 3301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 3361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 3421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 3481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 3541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 3601 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 3661 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 3721 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 3781 ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc 3841 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta 3901 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg 3961 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag 4021 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt 4081 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa 4141 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc 4201 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt 4261 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg 4321 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa 4381 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg 4441 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat 4501 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc 4561 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt 4621 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt 4681 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg 4741 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg 4801 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg 4861 aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 4921 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 4981 aattaatgta agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 5041 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 5101 tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 5161 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 5221 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct 5281 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg 5341 atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca 5401 acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt 5461 gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta 5521 tttttgatgg cgttggaatt agcttatcga ctgcacggtg caccaatgct tctggcgtca 5581 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt 5641 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg 5701 gcaaatattc tgaaatgagc tg //