LOCUS       sequence-175720-        7179 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 7179)
  AUTHORS   Violin JD, Zhang J, Tsien RY, Newton AC
  TITLE     A genetically encoded fluorescent reporter reveals oscillatory 
            phosphorylation by protein kinase C.
  JOURNAL   J Cell Biol. 2003 Jun 9. 161(5):899-909.
  PUBMED    12782683
REFERENCE   2  (bases 1 to 7179)
  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"; journalName: "J Cell 
            Biol. 2003 Jun 9. 161(5):899-909."
COMMENT     Sequence Label: DAGR
FEATURES             Location/Qualifiers
     source          1..7179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        155..534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        535..738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     689..709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     783..802
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        783..801
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      816..825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(822..824,825..827,828..1538)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(1920..1922,1923..2636)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        complement(2652..2670)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2653..2670)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2690..2707)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2696..2920
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2966..3394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3053..3072)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3263..3284
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3403..3423)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3408..3737
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3588..3723
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3650..3669
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3804..4598
                     /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(3858..3877)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4468..4487
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4772..4893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4809..4828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4863..4882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4942..4958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4942..4958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4955..4977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4966..4982
                     /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(4990..4996,4997..5014,5015..5020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5035..5056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6103..6894,6895..6963))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6726..6745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6964..7068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7143..7162)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
       61 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
      121 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      181 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      241 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      301 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      361 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      421 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      481 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      541 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      601 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      661 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      721 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
      781 attaatacga ctcactatag ggagacccaa gcttcgccac catggtgagc aagggcgagg
      841 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      901 ggttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
      961 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1021 ggggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1081 ccgccatgcc cgaaggctac gtccaggagc gtaccatctt cttcaaggac gacggcaact
     1141 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1201 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1261 tcagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag gcccacttca
     1321 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1381 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1441 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1501 ccgccgggat cactcacggc atggacgagc tgtacaaggc tggtaccgga ggccacaaat
     1561 tcaccgcccg cttcttcaag cagcccacct tctgcagcca ctgcaccgac ttcatttggg
     1621 gcttcgggaa acagggattc cagtgtcaag tctgctgctt tgttgtacac aagcgctgcc
     1681 atgaattcgt cacgttctcc tgccctggtg cagacaaggg cccggcctct gatgacccac
     1741 ggagcaaaca caagtttaag atccacacct actccagccc taccttctgt gaccactgtg
     1801 gatcactgct gtatgggctc atccaccagg ggatgaaatg cgacacctgt atgatgaatg
     1861 tccacaagcg ctgcgtgatg aacgtcccca gcctctgtgg cggaggtctc gaggcactgg
     1921 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1981 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2041 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2101 tgaccacctt cggctacggc ctgaagtgct tcgcccgcta ccccgaccac atgaagcagc
     2161 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2221 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2281 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2341 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2401 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2461 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2521 tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2581 tggagttcgt gaccgccgcc gggatcactc acggcatgga cgagctgtac aagtaatcta
     2641 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     2701 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2761 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2821 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2881 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2941 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3001 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3061 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3121 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3181 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3241 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3301 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3361 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3421 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3481 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3541 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3601 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3661 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3721 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3781 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3841 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3901 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3961 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4021 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4081 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4141 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4201 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4261 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4321 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4381 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4441 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4501 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4561 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4621 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4681 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4741 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4801 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4861 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4921 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4981 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5041 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5101 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6241 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7081 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     7141 agtacaatct gctctgatgc cgcatagtta agccagtat
//
