LOCUS       sequence-245654-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct for fusion expression of GeNL calcium reporter and 
            neomycin/G418 resistance gene by a bidirectional promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7901)
  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 7901)
  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-GeNL-Ca
FEATURES             Location/Qualifiers
     source          1..7901
                     /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             4162..4239
                     /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"
     CDS             4564..4590
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     rep_origin      complement(5615..6203)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5695..5714)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6374..7165,7166..7234))
                     /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     6997..7016
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7235..7339)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7376..7879
                     /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 gcatggtctc taagggggaa gaagacaata tggcctcgct gcccgctacg
     2881 catgaactcc acatttttgg ttccatcaac ggtgtggatt tcgatatggt tggccaaggg
     2941 accggcaacc caaatgacgg ctatgaagag ttaaatctca agagcacgaa aggggatcta
     3001 cagtttagtc cgtggatact tgtgcctcac atcggatacg gatttcacca atacctgccc
     3061 taccctgacg gaatgagccc tttccaggcc gctatggttg atggaagtgg ttaccaggtc
     3121 catcggacga tgcaatttga ggatggggcg agcctgacgg tgaattacag atacacctat
     3181 gaggggagcc atataaaggg cgaagcccaa gtcaagggga ccgggtttcc agcggacgga
     3241 ccagtgatga caaatagttt gactgccgcg gattggtgtc gatccaaaaa gacatatcct
     3301 aacgacaaga cgataatcag tacgttcaaa tggtcctaca cgacgggtaa tggtaaacga
     3361 tataggagca cggcgcggac tacctatacc ttcgcaaaac cgatggctgc aaattacttg
     3421 aagaaccagc ctatgtatgt cttccgcaag accgagctga agcacagcaa gacggaactc
     3481 aattttaagg aatggcagaa agcctttacg gggttcgagg acttcgtggg cgactggcgg
     3541 cagacggctg gctataacct cgaccaggtg ctggagcagg gcggtgtgtc ctcgcttttc
     3601 cagaacctgg gcgtctccgt gaccccaatc cagcgcatcg tcctgagcgg cgagaacggc
     3661 ctgaagatcg acatccacgt catcattccc tacgaggggc cttggatgca cgatcagctc
     3721 actgaggaac aaattgcaga gttcaaggaa gcgttctcac tttttgataa ggatggggat
     3781 gggactataa cgaccagaga gctaggaacc gtaatgcgtt cgctgggcca aaaccctact
     3841 gaggcggagt tgcaagatat gattaacgag gtggacgctg atggcaatgg aacgatttat
     3901 tttcccgagt ttttgacgat gatggcccgg aagatgaagg ataccgactc agaggaggag
     3961 atccgtgagg catttcgggt gttcgacaaa gacggaaacg gatacatctc cgctgcccag
     4021 ctcagacacg tcatgacgaa cttgggcgtg aaactgacgg acgaggaggt cgatgagatg
     4081 attcgggaag cggacataga cggtgatggg caggtgaact acgaggagtt tgagcagatg
     4141 atgactgcca agggggggtc gaagcgccgt tggaagaaga acttcattgc ggtctccgcg
     4201 gccaaccgat tcaagaaaat cagttcttcc ggcgcgcttg agttgctgag cggggaccaa
     4261 atgggccaga tcgagaagat cttcaaggtt gtgtaccccg tggacgatca ccacttcaag
     4321 gtgatactgc actacgggac gctggtcatc gacggtgtga cgccgaacat gattgactac
     4381 ttcggccgtc cttacgaggg cattgccgtg ttcgacggca agaagatcac ggtgaccggc
     4441 accctgtgga acggcaataa gatcattgac gagcggctga tcaacccgga tggctcgctg
     4501 ttgttccgcg tgaccatcaa cggggtaacc ggctggcgcc tgtgcgagcg catcctggcc
     4561 ggatacccct acgacgtgcc cgactacgcg ggaagcggca ggcctacgcg tgagtcgcga
     4621 tgagctcgaa agatccaaga gagacgagta gagatttttt ttttgggatt gatgtttgtc
     4681 gttctttgag ttgtcgtcga gttacgcctt ttgtaagaat gttccgcagg agaggaggag
     4741 gatgggcatg agtgagggtg agagggcttg cccgcttttt tttttaaaaa cgctgaagac
     4801 gtggttgtca aacaaacccc ccatagaaac gattttgtta cggtgcggtc cagacgtcac
     4861 ttgaatggct ccgcggaaag gccagggagg gaagggggga gggaggaaac atgaaacatg
     4921 ttgaacggct caacagggtt tgggggacaa gagaggtagc gccctgatgg actgctccct
     4981 cccctccttt ccctcaatgt ctcattcatc catgcttccc ccttctctct ctcccctccg
     5041 ttccatcccc cgcgggcgtg gtagtggcgt gatgggatcc actaaaatgt acgtgtaaga
     5101 aaagccggtg agcttacgct tttgtgaaag tgggagtacg agtgttgtgt gtgtgtgtag
     5161 tggtttcaga ccccagacag aggcgaagca gaaaaagcag acgatgaaga cgacgaagaa
     5221 atgagcagtc tatttttatc gtggaaacag aagaggtgat atcgtctcgt tctttgttat
     5281 cacctacccc gcgtgcatgt acatgcagcc tttttatttt gtaatctttc ccgaaaaatc
     5341 aaccgccacc tcccccccgc cttctctcac ccatcatctt ctcctgttta tcttctactt
     5401 tacactagat cgcatggcac atctccctcg caatccatcg gtgcaaccat catcgatccc
     5461 actcctccct ccctccctcc ctccctcctc gcatctcttc taagaaatcc gctagtgttt
     5521 aaaccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5581 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5641 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5701 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5761 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5821 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5881 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5941 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6001 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6061 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6121 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6181 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6241 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6301 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6361 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6421 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6481 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6541 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6601 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6661 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6721 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6781 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6841 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6901 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6961 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7021 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7081 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7141 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7201 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7261 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7321 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc ttttcatcac
     7381 gtgctataaa aataattata atttaaattt tttaatataa atatataaat taaaaataga
     7441 aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct
     7501 agggggatcg ccaacaaata ctacctttta tcttgtactt cctgctctca ggtattaatg
     7561 ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt gattttacat
     7621 tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat
     7681 atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca
     7741 ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa aacataaaaa
     7801 taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt
     7861 aagttacagg caagcgatcc gtctaagaaa ccggttaagg c
//
