LOCUS       sequence-210241-        7571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct for expression of a calcium sensitive luciferase reporter,
            GFP-aequorin, and hygromycin resistance gene by a bidirectional 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7571)
  AUTHORS   Poliner E, Cummings C, Newton L, Farre EM
  TITLE     Identification of circadian rhythms in Nannochloropsis species using
            bioluminescence reporter lines.
  JOURNAL   Plant J. 2019 Jul;99(1):112-127. doi: 10.1111/tpj.14314. Epub 2019 
            Apr 19.
  PUBMED    30883973
REFERENCE   2  (bases 1 to 7571)
  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"; doi: "10.1111/tpj.14314";
            journalName: "Plant J"; date: "2019-07"; volume: "99"; issue: "1"; 
            pages: "112-127"
COMMENT     Sequence Label: pNOC-stacked-GFP-aequorin
FEATURES             Location/Qualifiers
     source          1..7571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(803..1597)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(803..1597)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(914..933)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1524..1543
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1823..1834)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(2833..2835,2836..2838,2839..3549)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2878..2899)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3139..3158)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3486..3507
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(5285..5873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5365..5384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6044..6835,6836..6904))
                     /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     6667..6686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6905..7009)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7046..7549
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 taatggatgg gaagtcggta agtgaaaagg gctttgtgta aggtggatgt caaattttgc
       61 caacccgtgc ctctgtgttc gcagtgtagc aaggaagccc acaccaccgc ctcatattca
      121 aatcgaaatc atcgaagaaa tcggcgagga tctcatgccc caaagctgtc atcaaatgtg
      181 tcccaccaca cgaagatgcc gagtaaatga cgtgagttgt cctatgattg gcaggatgtg
      241 gcatttaaag tccaaaaggc cctgcttggt cggtcacctt ttgctcgcct ccccctcccg
      301 tcacacccta aacacgaatg cagaagaaaa tcatgtgggg aagcaacgat ttagaaatgg
      361 gaatggaggc tcttcacttc ctgaatctga ccaagctggc ggacacaacc gtagagggat
      421 ctttgacctg catcaataca ccgacatgtt aataaatcga gtaccaaaga aaagctactg
      481 aagtcagttt cggattgtgt tgtagaatct tttgagtcgt cactggaatg ctatgctcgc
      541 ttcaatcgtc gattagcttt gtaaattatt tctggtggca aggcggggtt caagtagcaa
      601 gtgggggcat ggcagcaccc ctccgagcct cgttcgttca ggtgtttttg ggacctttcc
      661 ctcccttcct gtcttcagac gcttttattt tcctttccgt tttctactag tgacaaaaaa
      721 agggcacaca ctctttactc gctcacaccc tcgtactcgg acacgcgctg ttaccgactc
      781 tggtacctca agcgcttcta gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
      841 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
      901 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
      961 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     1021 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc
     1081 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     1141 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     1201 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     1261 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     1321 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     1381 tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca
     1441 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     1501 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     1561 ccggagaacc tgcgtgcaat ccatcttgtt caatcatggt cagctgctcg agggttgcgt
     1621 gtgtatctgt gtgcagtgga tattgttacc gagtttggtg agcgtgagtc cgttagtgcc
     1681 ctggtggtgg tggattagga gagtgggtga ctcggtgtcc atggctttct tcgctcatta
     1741 taggagggga aaggaatgag ggagggtggg gagaccgcgt ctgttgttga ccaccgattt
     1801 acttcttgcc tcccttcccc tccctcccct caatccgtac gacacaaata gtagccgagt
     1861 gtctgctgca gagcgcatga ttagtgtggt agacaacgag ggagggaagg atgtacaggg
     1921 catggcacgg agaagcgatg gtggccagga agaggagagg tcgtgagaac aggatgtgtt
     1981 gcgaatggat aaaaacagaa agcgatggct ctgggcttcg aaagcagggg acattaggac
     2041 gtgtagacca tctcgacgga tccctctgta tctctgttgt gcgtgaatgt tttctgtgca
     2101 cgtgtagtgt gtgagagtag aacccgggaa ctcgaacaga gaaaagcatg ggtggctgtg
     2161 gtgtggaggc ttcgttccca ccacatgccc ttctccttcg cctcgcctct ccctgccttc
     2221 ttccacgcac ccttgcgccc ctcgttctca atacctggct cacttccacc attcaaacaa
     2281 ccatcacgat acaggcattt atctatcgtt gaagacttct tcctccggta gatcttagcc
     2341 aaggtaagaa gaggggcatg cagcaaggag aaagaaatga tgcatgatga ggaatagaag
     2401 gggaggaggg agggatatga tgggaagcga aagcgcatat tctggtggtc tgctgcctga
     2461 tggggagtct agctgtgaca ctgaggacgg tggctgctgg tggctgcggg cgctgccttg
     2521 gtgatcaatg ggagtaaagg gagggaagga ggtagcgtga acggatgacg cggagaagtt
     2581 taggggtctc tttacgtatc gcccctgccg cccgcctctc tgcgataaat gtgcctgtta
     2641 ccctgcagcc tctattcttc actgtgttcc tgttttccaa cagcctctat tcttccctgt
     2701 cttttgttgc agtggcgtca tcctctcttt gccccagtcg tcgttctctc gactcactca
     2761 ctccccccct ccttccctcc ctccatccac agaatcgatt ggcgcgccct cgttaaccaa
     2821 ttggggagcg gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2881 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2941 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3001 ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcag ccgctacccc
     3061 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3121 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3181 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3241 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3301 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3361 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3421 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3481 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag
     3541 ctgtacaagg gaagcggcag gcctacgcgt atgtcgggcg gtagcggaag tgggggccag
     3601 agcgggggga gtggctccgg aggacagtcc ggcgggagcg ggtcaggcgg tcaatccggc
     3661 ggtagcgggt cgggggggca atcgggaggc tccggttctg ggggccagac gagcaagtac
     3721 gccgtgaaat tggagcccga tttcgagaac cctcgatgga tcgggcgtca taagcacatg
     3781 tttaacttct tggacgtgaa tcaaaatggt aagattagct tagacgagat ggtttacaag
     3841 gcaagtgaca ttgttattaa caatctaggc gcaaccccgg agcaagcgaa gcgtcacaag
     3901 gaagcggtgg aggcgttctt cggcggagct ggcatgaagt acggggtgga gaccgagtgg
     3961 cccgagtaca tcaaggggtg gaagaagttg gcccaaactg aactggacaa gttcgccaaa
     4021 aaccaggtca cactcatacg gctctggggg gacgcgctgc ttgatattat tgacaaggat
     4081 cagaatggcg ctattaccct ggacgagtgg aagaagtaca ctcaatcggc tgggattatc
     4141 cagagcgccg aggattgcga ggagaccttt aaggtctgcg atcttgacga ttccgggcgt
     4201 ttggatgcag acgaaatgac ccgacagcat atcggttttt ggtacacgat ggaccccgcc
     4261 tgtgagaagc tctacggcgg cgcggtccct tgagctcgaa agatccaaga gagacgagta
     4321 gagatttttt ttttgggatt gatgtttgtc gttctttgag ttgtcgtcga gttacgcctt
     4381 ttgtaagaat gttccgcagg agaggaggag gatgggcatg agtgagggtg agagggcttg
     4441 cccgcttttt tttttaaaaa cgctgaagac gtggttgtca aacaaacccc ccatagaaac
     4501 gattttgtta cggtgcggtc cagacgtcac ttgaatggct ccgcggaaag gccagggagg
     4561 gaagggggga gggaggaaac atgaaacatg ttgaacggct caacagggtt tgggggacaa
     4621 gagaggtagc gccctgatgg actgctccct cccctccttt ccctcaatgt ctcattcatc
     4681 catgcttccc ccttctctct ctcccctccg ttccatcccc cgcgggcgtg gtagtggcgt
     4741 gatgggatcc actaaaatgt acgtgtaaga aaagccggtg agcttacgct tttgtgaaag
     4801 tgggagtacg agtgttgtgt gtgtgtgtag tggtttcaga ccccagacag aggcgaagca
     4861 gaaaaagcag acgatgaaga cgacgaagaa atgagcagtc tatttttatc gtggaaacag
     4921 aagaggtgat atcgtctcgt tctttgttat cacctacccc gcgtgcatgt acatgcagcc
     4981 tttttatttt gtaatctttc ccgaaaaatc aaccgccacc tcccccccgc cttctctcac
     5041 ccatcatctt ctcctgttta tcttctactt tacactagat cgcatggcac atctccctcg
     5101 caatccatcg gtgcaaccat catcgatccc actcctccct ccctccctcc ctccctcctc
     5161 gcatctcttc taagaaatcc gctagtgttt aaaccacaga atcaggggat aacgcaggaa
     5221 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5281 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5341 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5401 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5461 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5521 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5581 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5641 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5701 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5761 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5821 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5881 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5941 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6001 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6061 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6121 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6181 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6241 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6301 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6361 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6421 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6481 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6541 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6601 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6661 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6721 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6781 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6841 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6901 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6961 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7021 gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata atttaaattt
     7081 tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag
     7141 tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta
     7201 tcttgtactt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag
     7261 accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta
     7321 tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt
     7381 tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac
     7441 tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta
     7501 ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc gtctaagaaa
     7561 ccggttaagg c
//
