LOCUS       sequence-10189-e        5998 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 5998)
  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 5998)
  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: peCALWY-4
FEATURES             Location/Qualifiers
     source          1..5998
                     /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="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALFKDPNEKRDHMVLL
                     EFLTAAGITLGMDELYK"
     CDS             join(1944..1946,1947..1949,1950..2660)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITLGMDELYK"
     polyA_signal    2786..2907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2823..2842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2877..2896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2914..3369)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3051..3072)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3263..3282
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3396..3500
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3502..3859
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3502..3693
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3525..3545)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3710..3845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3894..4688
                     /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             3894..4688
                     /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(3948..3967)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4558..4577
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4876..4895)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4920..4967
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5296..5884
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5785..5804
                     /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 ctacggcaag 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 ccgccctgtt caaagacccc aacgagaagc
     1261 gcgatcacat ggtcctgctg gagttcctga ccgccgccgg gatcactctc ggcatggacg
     1321 agctgtacaa gtccggaatg ccgaagcacg agttctctgt ggacatgacc tgtggaggct
     1381 gtgctgaagc tgtctctcgg gtcctcaata agcttggagg agttaagtat gacattgacc
     1441 tgcccaacaa gaaggtctgc attgaatctg agcacagcat ggacactctg cttgcaaccc
     1501 tgaagaaaac aggaaagact gtttcctacc ttggccttga gctcattcgt ggcggatccg
     1561 gcggaagcgg cggatccggc ggtagcggcg gatccggcgg ctccggcgga tccggcggca
     1621 gcggcggatc cggtggaagc ggtggatccg gtggtagcgg tggatccggt ggaagcggtg
     1681 gatccggtgg tagcggtgga tccgggggtc cgcggatgca gggcacatgc agtaccactc
     1741 tgattgccat tgccggcatg acctgtgcat ccagtgtcca ttccattgaa ggcatgatct
     1801 cccaactgga aggggtgcag caaatatcgg tgtctttggc cgaagggact gcaacagttc
     1861 tttataatcc cgctgtaatt agcccagaag aactcagagc tgctatagaa gacatgggat
     1921 ttgaggcttc agtctcggta cccatggtga gcaagggcga ggagctgttc accggggtgg
     1981 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2041 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2101 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg atgtgcttcg
     2161 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2221 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2281 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2341 aggacggcaa catcctgggg cacaagcttg agtacaacta caacagccac aacgtctata
     2401 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2461 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2521 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgttc aaagacccca
     2581 acgagaagcg cgatcacatg gtcctgctgg agttcctgac cgccgccggg atcactctcg
     2641 gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac
     2701 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2761 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2821 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2881 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     2941 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3001 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     3061 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     3121 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3181 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3241 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3301 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3361 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3421 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3481 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3541 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3601 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3661 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3721 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3781 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     3841 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     3901 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3961 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4021 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     4081 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4141 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4201 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4261 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4321 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4381 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4441 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4501 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4561 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4621 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4681 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4741 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4801 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4861 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     4921 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     4981 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     5041 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     5101 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     5161 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5221 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5281 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5341 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5401 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5461 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5521 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5581 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5641 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5701 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5761 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5821 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5881 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5941 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
