LOCUS       sequence-129209-        6668 bp    DNA     circular SYN 14-APR-2025
DEFINITION  expresses ER-eZinCh-2 Zn2+ FRET sensor in the ER of mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6668)
  AUTHORS   Hessels AM, Chabosseau P, Bakker MH, Engelen W, Rutter GA, Taylor 
            KM, Merkx M
  TITLE     eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic
            and Intraorganelle Zn(2+) Imaging.
  JOURNAL   ACS Chem Biol. 2015 Sep 18;10(9):2126-34. doi: 
            10.1021/acschembio.5b00211. Epub 2015 Jul 20.
  PUBMED    26151333
REFERENCE   2  (bases 1 to 6668)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.5b00211"; journalName: "ACS Chem Biol"; date: 
            "2015-09-18- 18"; volume: "10"; issue: "9"; pages: "2126-34"
COMMENT     Sequence Label: pER-eZinCh-2
FEATURES             Location/Qualifiers
     source          1..6668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     901..972
                     /codon_start=1
                     /product="signal sequence of human preproinsulin"
                     /label=insulin signal sequence
                     /translation="MALWMRLLPLLALLALWGPDPAAA"
     CDS             join(979..981,982..984,985..1695)
                     /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(1897..1899,1900..1902,1903..2613)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2663..2680)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2669..2893
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2939..3367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3026..3045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3236..3257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3376..3396)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3381..3710
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3561..3696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3623..3642
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3777..4571
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3831..3850)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4441..4460
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4745..4866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4782..4801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4836..4855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4915..4931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4915..4931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4928..4950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4939..4955
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4963..4969,4970..4987,4988..4993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5008..5029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5146..5163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5317..5902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5397..5416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg gggacctgac
      961 ccagccgcag ccgaattcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1021 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1081 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggtaagctg
     1141 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
     1201 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1261 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1321 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1381 ggcaacatcc tggggcacaa gctggagtac aacgccatca gcgacaacgt ctatatcacc
     1441 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1501 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1561 ctgctgcccg acaaccacta cctgagcacc cagtcccacc tgtgcaaaga ccccaacgag
     1621 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1681 gacgagctgt acaagtccgg aggcggcgag ctcattcgtg gcggatccgg cggaagcggc
     1741 ggatccggcg gtagcggcgg atccggcggc tccggcggat ccggcggcag cggcggatcc
     1801 ggtggaagcg gtggatccgg tggtagcggt ggatccggtg gaagcggtgg atccggtggt
     1861 agcggtggat ccgggggtcc gcggggctcg gtacccatgg tgagcaaggg cgaggagctg
     1921 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1981 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2041 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
     2101 ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2161 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2221 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2281 atcgacttca aggaggacgg caacatcctg gggcacaagc ttgagtacaa ctacaacagc
     2341 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2401 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2461 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtcccacctg
     2521 tgcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2581 gggatcactc tcggcatgga cgagctgtac aaggtcgaca aggacgagct gtaagggccc
     2641 taagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2701 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2761 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2821 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2881 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     2941 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3001 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3061 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3121 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3181 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3241 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3301 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3361 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3421 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3481 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3541 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3601 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3661 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3721 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3781 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3841 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3901 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3961 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4021 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4081 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4141 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4201 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4261 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4321 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4381 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4441 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4501 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4561 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4621 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4681 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4741 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4801 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4861 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4921 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4981 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5041 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5101 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5161 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5221 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5281 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5341 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5401 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5461 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5521 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5581 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5641 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5701 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5761 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5821 ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac
     5881 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5941 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6001 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6061 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6121 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6181 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6241 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6301 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6361 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6421 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6481 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6541 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6601 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6661 gcgaccga
//
