LOCUS Exported 6003 bp ds-DNA circular SYN 03-MAR-2022 DEFINITION for bacterial expression of recombinant ratiometric pHluorin2. ACCESSION . VERSION . KEYWORDS RpHLuorin2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6003) TITLE Fluorescence lifetime imaging of pH along the secretory pathway JOURNAL Unpublished REFERENCE 2 (bases 1 to 6003) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 3, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6003 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 845..864 /label=LacI-R /note="LacI, reverse primer" promoter complement(884..961) /gene="lacI" /label=lacI promoter primer_bind 1167..1186 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1270..1289 /label=T7 /note="T7 promoter, forward primer" promoter 1270..1288 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1289..1313 /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)." RBS 1328..1350 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1369..1386 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1396..1413 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 2137..2154 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2207..2225) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2221..2268 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2305..2760 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2392..2411) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2602..2623 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2852..3667) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 3575..3594 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3659..3678 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3789..4377 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4278..4297 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4531..4548 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4563..4702 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4628..4647) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4788..4810 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4804..4995) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5804..883) /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" ORIGIN 1 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 61 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 121 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 181 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 241 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 301 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 361 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 421 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 481 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 541 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 601 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 661 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 721 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 781 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 841 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 901 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 961 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 1021 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 1081 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 1141 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 1201 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 1261 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 1321 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat 1381 catcacagca gcggcctggt gccgcgcggc agccatatgt ctaaaggtga agaactgttc 1441 accggtgttg ttccgatcct ggttgaactg gacggtgacg ttaacggtca caaattctct 1501 gtttctggtg aaggtgaagg tgacgctacc tacggtaaac tgaccctgaa attcatctgc 1561 accaccggta aactgccggt tccgtggccg accctggtta ccaccctgtc ttacggtgtt 1621 cagtgcttct ctcgttaccc ggaccacatg aaacagcacg acttcttcaa atctgctatg 1681 ccggaaggtt acgttcagga acgtaccatc ttcttcaaag acgacggtaa ctacaaaacc 1741 cgtgctgaag ttaaattcga aggtgacacc ctggttaacc gtatcgaact gaaaggtatc 1801 gacttcaaag aagacggtaa catcctgggt cacaaactgg aatacaacta caacgaacac 1861 ctggtttaca tcatggctga caaacagaaa aacggtacca aagctatctt ccaggttcac 1921 cacaacatcg aagacggtgg tgttcagctg gctgaccact accagcagaa caccccgatc 1981 ggtgacggtc cggttctgct gccggacaac cactacctgc acacccagtc tgctctgtct 2041 aaagacccga acgaaaaacg tgaccacatg gttctgctgg aattcgttac cgctgctggt 2101 atcacccacg gtatggacga actgtacaaa ctcgagcacc accaccacca ccactgagat 2161 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa 2221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga 2281 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg 2341 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 2401 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 2461 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 2521 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 2581 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 2641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 2701 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt 2761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 2821 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa 2881 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 2941 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 3001 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 3061 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat 3121 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 3181 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 3241 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 3301 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 3361 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 3421 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 3481 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 3541 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 3601 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat 3661 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc 3721 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 3781 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 3841 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 3901 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 3961 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4021 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4081 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4141 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4201 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4261 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4321 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4381 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 4441 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 4501 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca 4621 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct 4681 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg 4741 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 4801 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc 4861 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag 4921 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt 4981 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat 5041 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt 5101 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat 5161 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca 5221 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc 5281 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt 5341 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt 5401 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg 5461 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc 5521 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat 5581 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac 5641 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc 5701 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc 5761 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca 5821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 5881 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat 5941 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 6001 gca //