LOCUS       sequence-374516-        5983 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of BLZinCh-P20, a dual read-out BRET-FRET sensor for 
            intracellular Zn2+ imaging, in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5983)
  AUTHORS   Michielsen CMS, van Aalen EA, Merkx M
  TITLE     Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum 
            and Intracellular Free Zn(2).
  JOURNAL   ACS Chem Biol. 2022 Jun 17;17(6):1567-1576. doi: 
            10.1021/acschembio.2c00227. Epub 2022 May 25.
  PUBMED    35611686
REFERENCE   2  (bases 1 to 5983)
  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: 
            "10.1021/acschembio.2c00227"; journalName: "ACS Chem Biol"; date: 
            "2022-06-17- 17"; volume: "17"; issue: "6"; pages: "1567-1576"
COMMENT     Sequence Label: pCMV-BLZinCh-P20
FEATURES             Location/Qualifiers
     source          1..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..367
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        368..571
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     522..542
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1110..1112,1113..1115,1116..1826)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1920..1922,1923..1925,1926..2636)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    2774..2895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2811..2830)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2865..2884
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2902..3357)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3039..3060)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3251..3270
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3384..3488
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3490..3847
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3490..3681
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3513..3533)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3698..3833
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3882..4676
                     /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             3882..4676
                     /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(3936..3955)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4546..4565
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4864..4883)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4908..4955
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5284..5872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5773..5792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
       61 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      121 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      181 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      241 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      301 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      361 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      421 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      481 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      541 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgtgctag
      601 ccatggtatt tactcttgaa gattttgtcg gtgattggcg ccagaccgcc ggctataacc
      661 tggaccaagt gcttgaacag ggcggggtta gcagcctgtt tcaaaacctg ggggtgagtg
      721 tcacgccaat tcagcgcatc gttctgtcgg gagagaatgg tctgaaaatc gatatccacg
      781 tcattatccc gtacgaaggt ctttctggtg atcagatggg gcagatagaa aaaatattca
      841 aagtggtgta cccagtagac gatcatcact tcaaggttat actgcactat ggcaccctcg
      901 ttatcgatgg cgttactccg aatatgatcg attactttgg gcgtccttat gaaggtattg
      961 cggtgttcga cggtaaaaaa attacggtta ccgggacgct ctggaatggt aataaaatca
     1021 ttgatgagcg cttgataaac ccagatggca gccttctgtt cagagttacg ataaacgggg
     1081 ttacgggttg gcgactgtgc gaaagacata tggtgagcaa gggcgaagag ctgttcaccg
     1141 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1201 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1261 ccggtaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt
     1321 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1381 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1441 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1501 tcaaggagga cggcaacatc ctggggcaca agctggagta caacgccatc agcgacaacg
     1561 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     1621 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1681 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtcccac ctgtgcaaag
     1741 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1801 ctctcggcat ggacgagctg tacaagtccg gaggcggcga gctcccacct ccaccgccgc
     1861 ctccacctcc accgccaccc cctccacccc cgccacctcc tccgcggggc tcggtaccca
     1921 tggtgagcaa gggcgaagag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1981 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2041 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2101 tcgtgaccac cttcggctac ggcctgatgt gcttcgcccg ctaccccgac cacatgaagc
     2161 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2221 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2281 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2341 agcttgagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2401 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2461 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2521 acctgagcta ccagtcccac ctgtgcaaag accccaacga gaagcgcgat cacatggtcc
     2581 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaca
     2641 agcggccgca ctcgagctaa gactctagat cataatcagc cataccacat ttgtagaggt
     2701 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     2761 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2821 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2881 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2941 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3001 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     3061 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3121 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     3181 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3241 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3301 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     3361 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3421 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3481 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3661 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3721 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3781 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3841 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3901 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3961 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4021 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4081 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4141 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4201 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4261 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4321 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4381 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     4441 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4501 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4561 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4621 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4681 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4741 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4801 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     4861 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     4921 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     4981 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5041 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5101 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     5161 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5221 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5281 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5341 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5401 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5461 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5521 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5581 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5641 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5701 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5761 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5821 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5881 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5941 tgcgttatcc cctgattctg tggataaccg tattaccgcc atg
//
