LOCUS       sequence-365034-        6043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of BLZinCh-P40, 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 6043)
  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 6043)
  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-P40
FEATURES             Location/Qualifiers
     source          1..6043
                     /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(1977..1979,1980..1982,1983..2693)
                     /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    2831..2952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2868..2887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2922..2941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2959..3414)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3117)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3308..3327
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3441..3545
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3547..3904
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3547..3738
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3570..3590)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3755..3890
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3939..4733
                     /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             3939..4733
                     /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(3993..4012)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4603..4622
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4921..4940)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4965..5012
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5341..5929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5830..5849
                     /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 gccaccccct ccacccccgc cacctcctcc accgccacca ccgccccctc
     1921 cacctcctcc accgccccca ccgcccccgc cacctcctcc gcggggctcg gtacccatgg
     1981 tgagcaaggg cgaagagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2041 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2101 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2161 tgaccacctt cggctacggc ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc
     2221 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2281 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2341 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2401 ttgagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2461 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2521 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2581 tgagctacca gtcccacctg tgcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2641 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtacaagc
     2701 ggccgcactc gagctaagac tctagatcat aatcagccat accacatttg tagaggtttt
     2761 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2821 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2881 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2941 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3001 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3061 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3121 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3181 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3241 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3301 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3361 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3421 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3481 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3541 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3601 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3661 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3721 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3781 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3841 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3901 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3961 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4021 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4081 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4141 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4201 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4261 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4321 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4381 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4441 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4501 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4561 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4621 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4681 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4741 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4801 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4861 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4921 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     4981 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5041 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5101 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5161 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5221 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5281 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5341 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5401 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5461 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     5521 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5581 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5641 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5701 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5761 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5821 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5881 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5941 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6001 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
