LOCUS       sequence-374523-        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"
     CDS             complement(join(175..885,886..888,889..891))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGXXCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(1430..1633)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1459..1479)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1634..1937
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2112..2700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2192..2211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3029..3076
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3101..3120
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3308..4102)
                     /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(3308..4102)
                     /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(3419..3438)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4029..4048
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4137..4494)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4151..4286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4303..4494
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4451..4471
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(4496..4600)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4627..5082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4714..4733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4924..4945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5089..5210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5100..5119)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5154..5173
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5348..5599)
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKXNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSHLCKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
ORIGIN
        1 ctcttcgccc ttgctcacca tgggtaccga gccccgcgga ggaggtggcg ggggcggtgg
       61 gggcggtgga ggaggtggag ggggcggtgg tggcggtgga ggaggtggcg ggggtggagg
      121 gggtggcggt ggaggtggag gcggcggtgg aggtgggagc tcgccgcctc cggacttgta
      181 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg
      241 atcgcgcttc tcgttggggt ctttgcacag gtgggactgg gtgctcaggt agtggttgtc
      301 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg
      361 cacgctgccg tcctcgatgt tgtggcggat cttgaagttg gccttgatgc cgttcttctg
      421 cttgtcggcg gtgatataga cgttgtcgct gatggcgttg tactccagct tgtgccccag
      481 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc
      541 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt
      601 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg
      661 gtcggggtag cgggcgaagc acwkcasgcc ccaggtcagg gtggtcacga gggtgggcca
      721 gggcacgggc agcttaccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc
      781 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc
      841 gaccaggatg ggcaccaccc cggtgaacag ctcttcgccc ttgctcacca tatgtctttc
      901 gcacagtcgc caacccgtaa ccccgtttat cgtaactctg aacagaaggc tgccatctgg
      961 gtttatcaag cgctcatcaa tgattttatt accattccag agcgtcccgg taaccgtaat
     1021 ttttttaccg tcgaacaccg caataccttc ataaggacgc ccaaagtaat cgatcatatt
     1081 cggagtaacg ccatcgataa cgagggtgcc atagtgcagt ataaccttga agtgatgatc
     1141 gtctactggg tacaccactt tgaatatttt ttctatctgc cccatctgat caccagaaag
     1201 accttcgtac gggataatga cgtggatatc gattttcaga ccattctctc ccgacagaac
     1261 gatgcgctga attggcgtga cactcacccc caggttttga aacaggctgc taaccccgcc
     1321 ctgttcaagc acttggtcca ggttatagcc ggcggtctgg cgccaatcac cgacaaaatc
     1381 ttcaagagta aataccatgg ctagcacgga tctgacggtt cactaaacca gctctgctta
     1441 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
     1501 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
     1561 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
     1621 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
     1681 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     1741 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     1801 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
     1861 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     1921 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     1981 attactatta ataactaatg catggcggta atacggttat ccacagaatc aggggataac
     2041 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2101 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2161 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2221 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2281 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2341 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2401 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2461 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2521 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2581 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2641 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2701 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2761 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2821 tgaagtttta aatcaatcta aagtatatat gagtaacctg aggctatggc agggcctgcc
     2881 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg gggtggggaa
     2941 aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc gacagagtgc
     3001 cagccctggg accgaacccc gcgtttatga acaaacgacc caacaccgtg cgttttattc
     3061 tgtcttttta ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca
     3121 gttagcctcc ccctagggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
     3181 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
     3241 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga
     3301 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     3361 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     3421 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     3481 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     3541 gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg
     3601 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     3661 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     3721 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     3781 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     3841 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     3901 atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     3961 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     4021 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     4081 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcgatctttg
     4141 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag
     4201 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     4261 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     4321 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     4381 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     4441 gactttccac accctaactg acacacattc cacagctggt tctttccgcc tcaggactct
     4501 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4561 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4621 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4681 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4741 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4801 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4861 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4921 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4981 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5041 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttacgcctta agatacattg
     5101 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     5161 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     5221 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     5281 aaaacctcta caaatgtggt atggctgatt atgatctaga gtcttagctc gagtgcggcc
     5341 gcttgtactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
     5401 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgca caggtgggac tggtagctca
     5461 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     5521 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcrccttga
     5581 tgccgttctt ctgcttgtcg gcsatgatat agacgttgtg gctgttgtag ttgtactcaa
     5641 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     5701 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     5761 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     5821 gctgcttcat gtggtcgggg tagcgggcga agcacatcag gccgtagccg aaggtggtca
     5881 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     5941 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     6001 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cag
//
