LOCUS Exported 7200 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Cyan color bright luminescent calcium indicator with intermediate affinity. ACCESSION . VERSION . KEYWORDS CeNL(Ca2+)_19u-pcDNA3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7200) AUTHORS Hossain MN, Suzuki K, Iwano M, Matsuda T, Nagai T TITLE Bioluminescent Low-Affinity Ca(2+) Indicator for ER with Multicolor Calcium Imaging in Single Living Cells. JOURNAL ACS Chem Biol. 2018 Mar 9. doi: 10.1021/acschembio.7b01014. PUBMED 29494125 REFERENCE 2 (bases 1 to 7200) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7200 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 151..530 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 531..734 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 685..705 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 779..798 /label=T7 /note="T7 promoter, forward primer" promoter 779..797 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 830..1546 /codon_start=1 /product="enhanced monomeric variant of CFP (Goedhart et al., 2012)" /label=mTurquoise2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(875..896) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1136..1155) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1483..1504 /label=EGFP-C /note="EGFP, forward primer" CDS 2207..2284 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" promoter complement(2669..2687) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2670..2687) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2707..2724) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2713..2937 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2983..3411 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3070..3089) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3280..3301 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3420..3440) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3425..3754 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3605..3740 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3667..3686 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3821..4615 /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(3875..3894) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4485..4504 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4789..4910 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4826..4845) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4880..4899 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4959..4975) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4959..4975) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4972..4994) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4983..4999 /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(5007..5037) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5052..5073 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5190..5207) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5361..5949) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5441..5460) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6120..6980) /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 6743..6762 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6981..7085) /gene="bla" /label=AmpR promoter primer_bind complement(7160..7179) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 61 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 121 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 541 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 661 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 721 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta 781 atacgactca ctatagggag acccaagctt ggtaccgagc tcggatccga tggtgagcaa 841 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 901 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 961 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 1021 cctgtcctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt 1081 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 1141 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 1201 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 1261 caactacttt agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc 1321 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca 1381 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 1441 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 1501 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggta ccgtcttcac 1561 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct 1621 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca 1681 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta 1741 tgaaggtcca tggatgcatg accaactgac agaagagcag attgcagagt tcaaagaagc 1801 cttctcatta ttcgacaagg atggggacgg caccatcacc acaaaggaac ttggcaccgt 1861 tatgaggtcg cttggacaaa acccaacgga agcagaattg caggatatga tcaatgaagt 1921 cgatgctgat ggcaatggaa cgatttactt tcctgatttt cttactatga tggctagaaa 1981 aatgaaggac acagacagcg aagaggaaat ccgagaagca ttccgtgttt gggacaagga 2041 tgggaacggc tacatcagcg ctgctgattt acgtcacgtc atgacaaacc tcggggagaa 2101 gttaacagat gaagaagttg atgaaatgat aagggaagca gatatcgatg gtgaaggcca 2161 agtaaactat gaagagtttg tacaaatgat gacagcaaag ggggggaaga ggcgctggaa 2221 gaaaaacttc attgccgtca gcgctgccaa ccggttcaag aagatctcca gctccggggc 2281 actggagctc ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta 2341 ccctgtggat gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg 2401 ggttacgccg aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga 2461 cggcaaaaag atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg 2521 cctgatcaac cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg 2581 gcggctgtgc gaacgcattc tggcgtaaga attctgcaga tatccatcac actggcggcc 2641 gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct 2701 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 2761 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 2821 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 2881 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 2941 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 3001 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 3061 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 3121 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 3181 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 3241 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 3301 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 3361 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg 3421 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3481 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 3541 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 3601 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 3661 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg 3721 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat 3781 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt 3841 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 3901 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 3961 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 4021 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 4081 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 4141 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 4201 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 4261 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 4321 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 4381 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 4441 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 4501 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 4561 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 4621 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat 4681 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 4741 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt 4801 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 4861 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt 4921 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga 4981 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 5041 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5101 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5161 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5221 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5281 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5341 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5401 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5461 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5521 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5581 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5641 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5701 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5761 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 5821 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5881 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5941 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6001 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6061 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6121 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6181 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6241 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6301 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6361 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6421 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6481 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6541 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6601 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6661 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6721 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6781 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6841 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6901 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6961 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7021 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7081 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc 7141 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc //