LOCUS       sequence-11325-e        5587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5587)
  AUTHORS   Vinkenborg JL, Nicolson TJ, Bellomo EA, Koay MS, Rutter GA, Merkx M
  TITLE     Genetically encoded FRET sensors to monitor intracellular Zn(2+) 
            homeostasis.
  JOURNAL   Nat Methods. 2009 Oct;6(10):737-40. doi: 10.1038/nmeth.1368.
  PUBMED    19718032
REFERENCE   2  (bases 1 to 5587)
  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: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2009-10"; volume: "6"; issue: 
            "10"; pages: "737-40"
COMMENT     Sequence Label: peZinCh-1
FEATURES             Location/Qualifiers
     source          1..5587
                     /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"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(615..617,618..620,621..1331)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATHGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1533..1535,1536..1538,1539..2249)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATHGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    2375..2496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2412..2431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2466..2485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2503..2958)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2640..2661)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2852..2871
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2985..3089
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3091..3448
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3091..3282
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3114..3134)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3299..3434
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3483..4277
                     /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             3483..4277
                     /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(3537..3556)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4147..4166
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4465..4484)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4509..4556
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4885..5473
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5374..5393
                     /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 gctagcgccg
      601 ccaccatggg ccatatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      661 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      721 gcgatgccac ccacggcaag ctgaccctga agttcatctg caccaccggt aagctgcccg
      781 tgccctggcc caccctcgtg accaccctga cctggggcgt gcagtgcttc gcccgctacc
      841 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      901 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      961 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1021 acatcctggg gcacaagctg gagtacaacg ccatcagcga caacgtctat atcaccgccg
     1081 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca
     1141 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1201 tgcccgacaa ccactacctg agcacccagt ccgccctgtg caaagacccc aacgagaagc
     1261 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1321 agctgtacaa gtccggaggc ggcgagctca ttcgtggcgg atccggcgga agcggcggat
     1381 ccggcggtag cggcggatcc ggcggctccg gcggatccgg cggcagcggc ggatccggtg
     1441 gaagcggtgg atccggtggt agcggtggat ccggtggaag cggtggatcc ggtggtagcg
     1501 gtggatccgg gggtccgcgg ggctcggtac ccatggtgag caagggcgag gagctgttca
     1561 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1621 tgtccggcga gggcgagggc gatgccaccc acggcaagct gaccctgaag ttcatctgca
     1681 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctga
     1741 tgtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1801 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1861 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1921 acttcaagga ggacggcaac atcctggggc acaagcttga gtacaactac aacagccaca
     1981 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2041 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2101 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgtgca
     2161 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2221 tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag
     2281 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     2341 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     2401 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2461 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     2521 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     2581 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     2641 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     2701 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     2761 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     2821 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     2881 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     2941 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3001 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3061 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     3121 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3181 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3241 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3301 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3361 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     3421 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     3481 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3541 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3601 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3661 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3721 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3781 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3841 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3901 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3961 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     4021 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4081 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4141 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4201 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4261 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     4321 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4381 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4441 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     4501 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     4561 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     4621 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     4681 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     4741 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4801 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4861 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4921 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4981 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5041 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5101 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5161 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5221 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5281 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5341 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5401 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5461 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5521 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5581 catgcat
//
