LOCUS       sequence-214004-        7125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Orange color bright luminescent calcium indicator with intermediate 
            affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7125)
  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 7125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Biol. 2018 Mar 9. doi: 10.1021/acschembio.7b01014."
COMMENT     Sequence Label: OeNL(Ca2+)_18u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        162..541
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        542..745
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     696..716
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     790..809
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        790..808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             841..1494
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2143..2220
                     /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(2605..2623)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2606..2623)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2643..2660)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2649..2873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2919..3347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3006..3025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3216..3237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3356..3376)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3361..3690
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3541..3676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3603..3622
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3757..4551
                     /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(3811..3830)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4421..4440
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4725..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4762..4781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4816..4835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4895..4911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4895..4911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4908..4930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4919..4935
                     /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(join(4943..4949,4950..4967,4968..4973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4988..5009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5126..5143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5297..5885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5377..5396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6056..6847,6848..6916))
                     /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     6679..6698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6917..7021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7096..7115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
       61 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      121 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      181 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      241 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      301 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      361 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      421 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      481 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      541 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      601 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      661 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      721 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
      781 tatcgaaatt aatacgactc actataggga gacccaagct tggtaccgag ctcggatccg
      841 atggtgagcg tgatcaagcc cgagatgaag atgaggtact acatggacgg ctccgtcaat
      901 gggcatgagt tcacaatcga gggtgagggc acaggcagac cttacgaggg acatcaggag
      961 atgacactgc gcgtcacaat ggccgagggc gggccaatgc ctttcgcctt cgacctggtg
     1021 tcccacgtgt tctgttacgg ccacagagtt tttaccaagt acccagaaga gatcccagac
     1081 tatttcaagc aggcctttcc tgagggcctg tcctgggaga ggtccctgga gttcgaggac
     1141 ggcggctccg cctccgtgag cgcccacatc agcctgaggg gcaacacctt ctaccacaag
     1201 tccaagttca ccggcgtgaa cttccccgcc gacggcccca tcatgcagaa ccagagcgtg
     1261 gactgggagc cctccaccga gaagatcacc gccagcgacg gcgtgctgaa gggcgacgtg
     1321 accatgtacc tgaagctgga gggcggcggc aaccacaagt gccagttcaa gaccacctac
     1381 aaggccgcca aggagatcct ggagatgccc ggcgaccact acatcggcca caggctggtg
     1441 aggaagaccg agggcaacat caccgagcag gtggaggacg ccgtggccca ctccggtacc
     1501 gaagatttcg ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa
     1561 cagggaggtg tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg
     1621 attgtcctga gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa
     1681 ggtccatgga tgcatgacca actgacagaa gagcagattg cagagttcaa agaagccttc
     1741 tcattattcg acaaggatgg ggacggcacc atcaccacaa aggaacttgg caccgttatg
     1801 aggtcgcttg gacaaaaccc aacggaagca gaattgcagg atatgatcaa tgaagtcgat
     1861 gctgatggca atggaacgat ttactttcct gattttctta ctatgatggc tagaaaaatg
     1921 aaggacacag acagcgaaga ggaaatccga gaagcattcc gtgtttttga caaggatggg
     1981 aacggctaca tcagcgctgc tgatttacgt cacgtcatga caaacctcgg ggagaagtta
     2041 acagatgaag aagttgatga aatgataagg gaagcagata tcgatggtga aggccaagta
     2101 aactatgaag agtttgtaca aatgatgaca gcaaaggggg ggaagaggcg ctggaagaaa
     2161 aacttcattg ccgtcagcgc tgccaaccgg ttcaagaaga tctccagctc cggggcactg
     2221 gagctcctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct
     2281 gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt
     2341 acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc
     2401 aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg
     2461 atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg
     2521 ctgtgcgaac gcattctggc gtaagaattc tgcagatatc catcacactg gcggccgctc
     2581 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2641 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2701 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2761 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2821 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2881 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3361 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3421 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3481 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3541 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3601 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3661 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3721 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3781 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3841 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3901 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3961 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4021 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4081 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4141 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4201 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4261 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4321 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4381 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4441 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4501 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4561 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4621 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4681 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4741 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4801 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4861 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4921 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4981 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5041 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5101 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5161 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5221 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5281 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5341 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5401 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5461 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5521 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5581 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5641 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5701 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5761 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5821 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5881 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5941 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6001 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6061 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6661 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6721 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6781 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6841 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6901 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6961 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7021 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7081 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaag
//
