LOCUS       sequence-374515-        6013 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of BLZinCh-P30, 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 6013)
  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 6013)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-P30
FEATURES             Location/Qualifiers
     source          1..6013
                     /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(1947..1949,1950..1952,1953..2663)
                     /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    2801..2922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2838..2857)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2892..2911
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2929..3384)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3066..3087)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3278..3297
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3411..3515
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3517..3874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3517..3708
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3540..3560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3725..3860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3909..4703
                     /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             3909..4703
                     /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(3963..3982)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4573..4592
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4891..4910)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4935..4982
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5311..5899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5800..5819
                     /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 ccgcccccgc
     1921 cacctcctcc gcggggctcg gtacccatgg tgagcaaggg cgaagagctg ttcaccgggg
     1981 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2041 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2101 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgatgtgct
     2161 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2221 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2281 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2341 aggaggacgg caacatcctg gggcacaagc ttgagtacaa ctacaacagc cacaacgtct
     2401 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2461 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2521 gccccgtgct gctgcccgac aaccactacc tgagctacca gtcccacctg tgcaaagacc
     2581 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2641 tcggcatgga cgagctgtac aagtacaagc ggccgcactc gagctaagac tctagatcat
     2701 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2761 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2821 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2881 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     2941 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3001 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3061 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3121 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3181 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3241 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3301 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3361 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3481 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3541 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3601 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3661 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3721 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3781 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3841 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     3901 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3961 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4021 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4081 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4141 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4201 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4261 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4321 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4381 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4441 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4501 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4561 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4621 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4681 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4741 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4801 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4861 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     4921 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     4981 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     5041 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     5101 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     5161 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5221 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5281 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5341 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5401 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5461 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5521 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5581 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5641 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5701 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5761 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5821 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5881 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5941 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6001 taccgccatg cat
//
