LOCUS Exported 5489 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encoded a yellow fluorescent calcium ion indicator with inverted fluorescence response for expression in bacterial cells. ACCESSION . VERSION . KEYWORDS pTorPE-Y-GECO1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5489) AUTHORS Zhao Y, Abdelfattah AS, Zhao Y, Ruangkittisakul A, Ballanyi K, Campbell RE, Harrison DJ TITLE Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response. JOURNAL Integr Biol (Camb). 2014 May 19. PUBMED 24840546 REFERENCE 2 (bases 1 to 5489) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5489 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..285 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 208..227 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" RBS 327..338 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 493..510 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 514..546 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 601..657 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" CDS 1611..1625 /codon_start=1 /product="enterokinase recognition and cleavage site" /label=enterokinase site /translation="DDDDK" primer_bind complement(1890..1907) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 2060..2146 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2238..2265 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 2284..2375 /gene="bla" /label=AmpR promoter CDS 2376..3236 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2594..2613) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3407..3995 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3896..3915 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4149..4166 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4181..4321 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4247..4266) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4407..4429 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4585..5463) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" ORIGIN 1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 301 taacaggagg aattaaccat gggtttaaag aggagaaagg tcatgaacaa taacgatctc 361 tttcaggcat cacgtcggcg ttttctggca caactcggca gcttaaccga cgccgggacg 421 ctggggccgt cattgttaac gccgcgacgt gcgactgcgg cgcaagcggc gactgacgct 481 tctagaggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 541 atgggtcggg atctgtacga cgaagatgat aaggatctcg ccacaatggc cgactcatca 601 cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggccggct gagctcactc 661 gagggttata gtaccgagaa aattgtgcct gttccaaagc aagggatatt aaaaggaaag 721 gtcagcatgt acctattgct gaaggatggc ggtcgttatc attgccagtt tgatacggta 781 tataaagcta aatcagtgcc ctccaagatg ccaggacggc attttataca gcataagctc 841 cttagggagg accgctctga tgctaaaaac cataaatggc aattaacaga acatgcaatc 901 gcaggcatgg acgagctgta caagggaggt acaggcggga gtatggtgag caagggcgag 961 gggcaatcga agcatgggct aaaggaggag atgacggtga agtaccacat ggaaggttgc 1021 gttaatggtc acaaatttgt cattactgga gagggcattg gaaacccttt taagggtaag 1081 caaactgcaa atttgtgtgt gatagaagga ggcccgctgc cgttctcgga ggacatttta 1141 agtccgggtt ttaaatatgg tgaccggatt ttcacagagt acccacagga tattgtagat 1201 tacttcaaga actcatgtcc agagggctat acgtgggaaa ggagcttctt ctatgaggac 1261 ggagcggtct gtcgatgcaa cgcggacata acagtctctg aaaaggagaa ctgcatctat 1321 cacaaaagta tcttctatgg ggtgaatttt cccgccgacg gccccgtaat gaagaaaatg 1381 accactaatt gggaagctga cacgcgtgac caactgactg aagagcagat cgcagaattt 1441 aaagaggctt tctccctatt tgacaaggac ggggatggga cgatgacaac cgaggagctg 1501 gggacggtgc tgcggtctct ggggcagaac ccctcagaag cagagctgcg ggacatgatc 1561 aatgaagtag atgccgacgg tgacggcaca atcggcttcc ctgagttcct gacgatgatg 1621 acaagaaaaa tgaatgacac ggacagtgaa gaggaaatta gagaagcgtt ccgtgtgttt 1681 gataaggacg gcaatggcta catcggcgca gcagagcttc gccacgtgat gacaaacctt 1741 ggagagaagt taacagatga agagtttgat gaaatgatca gggtagcaga ctccgacggg 1801 gatggtcagg taaactacga agagtttgta caaatgatga cagcaaagtg aaagcttggc 1861 tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc 1921 ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg 1981 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga 2041 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 2101 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga 2161 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc 2221 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac 2281 tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 2341 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 2401 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 2461 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 2521 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 2581 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac 2641 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 2701 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 2761 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 2821 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 2881 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 2941 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 3001 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 3061 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 3121 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 3181 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 3241 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 3301 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 3361 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 3421 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 3481 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 3541 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 3601 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 3661 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 3721 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 3781 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 3841 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 3901 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 3961 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 4021 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 4081 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 4141 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc 4201 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 4261 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 4321 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 4381 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 4441 atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcata 4501 atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact ccgtcaagcc 4561 gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc 4621 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga 4681 gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt 4741 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct 4801 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg 4861 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc 4921 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg 4981 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg 5041 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg 5101 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc 5161 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta 5221 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg 5281 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat 5341 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc 5401 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc 5461 catacttttc atactcccgc cattcagag //