LOCUS       sequence-374521-        6073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of BLZinCh-P50, 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 6073)
  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 6073)
  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-P50
FEATURES             Location/Qualifiers
     source          1..6073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1107..1109,1110..1112,1113..1823)
                     /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(2007..2009,2010..2012,2013..2723)
                     /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    2861..2982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2898..2917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2952..2971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2989..3444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3338..3357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3471..3575
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3577..3934
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3577..3768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3600..3620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3785..3920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3969..4763
                     /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             3969..4763
                     /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(4023..4042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4633..4652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4951..4970)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4995..5042
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5371..5959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5860..5879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gtgctagcca
      601 tggtatttac tcttgaagat tttgtcggtg attggcgcca gaccgccggc tataacctgg
      661 accaagtgct tgaacagggc ggggttagca gcctgtttca aaacctgggg gtgagtgtca
      721 cgccaattca gcgcatcgtt ctgtcgggag agaatggtct gaaaatcgat atccacgtca
      781 ttatcccgta cgaaggtctt tctggtgatc agatggggca gatagaaaaa atattcaaag
      841 tggtgtaccc agtagacgat catcacttca aggttatact gcactatggc accctcgtta
      901 tcgatggcgt tactccgaat atgatcgatt actttgggcg tccttatgaa ggtattgcgg
      961 tgttcgacgg taaaaaaatt acggttaccg ggacgctctg gaatggtaat aaaatcattg
     1021 atgagcgctt gataaaccca gatggcagcc ttctgttcag agttacgata aacggggtta
     1081 cgggttggcg actgtgcgaa agacatatgg tgagcaaggg cgaagagctg ttcaccgggg
     1141 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1201 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1261 gtaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctggggc gtgcagtgct
     1321 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1381 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1441 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1501 aggaggacgg caacatcctg gggcacaagc tggagtacaa cgccatcagc gacaacgtct
     1561 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca
     1621 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1681 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtcccacctg tgcaaagacc
     1741 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1801 tcggcatgga cgagctgtac aagtccggag gcggcgagct cccacctcca ccgccgcctc
     1861 cacctccacc gccmcccccy ccmcccccsc cmccyccycc mccsccmccm ccscccccsc
     1921 cmccyccycc mccycccccm ccscccccyc cmccyccycc mccscccccm ccgcccccgc
     1981 cacctcctcc gcggggctcg gtacccatgg tgagcaaggg cgaagagctg ttcaccgggg
     2041 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2101 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2161 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgatgtgct
     2221 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2281 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2341 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2401 aggaggacgg caacatcctg gggcacaagc ttgagtacaa ctacaacagc cacaacgtct
     2461 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2521 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2581 gccccgtgct gctgcccgac aaccactacc tgagctacca gtcccacctg tgcaaagacc
     2641 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2701 tcggcatgga cgagctgtac aagtacaagc ggccgcactc gagctaagac tctagatcat
     2761 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2821 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2881 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2941 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     3001 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3061 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3421 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3541 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3601 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3661 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3721 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3781 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3841 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3901 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     3961 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4021 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4081 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4141 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4201 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4261 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4321 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4381 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4441 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4501 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4561 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4621 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4681 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4741 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4921 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     4981 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     5041 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     5101 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     5161 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     5221 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5281 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5341 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5401 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5461 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5521 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5581 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5641 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5701 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5761 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5821 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5881 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5941 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6001 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6061 taccgccatg cat
//
