LOCUS       sequence-67661-e        5282 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 5282)
  AUTHORS   Yasuda R, Harvey CD, Zhong H, Sobczyk A, van Aelst L, Svoboda K
  TITLE     Supersensitive Ras activation in dendrites and spines revealed by 
            two-photon fluorescence lifetime imaging.
  JOURNAL   Nat Neurosci. 2006 Feb . 9(2):283-91.
  PUBMED    16429133
REFERENCE   2  (bases 1 to 5282)
  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: "Nat 
            Neurosci. 2006 Feb . 9(2):283-91."
COMMENT     Sequence Label: mEGFP-HRas
FEATURES             Location/Qualifiers
     source          1..5282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1068..1077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1074..1076,1077..1079,1080..1790)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1119..1140)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1380..1399)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1727..1748
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1803..2372
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    2396..2517
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2407..2426)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2461..2480
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2698..3153
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2785..2804)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2995..3016
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3220..3239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3339..3361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3399..3417)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3485..3589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3590..3658,3659..4450)
                     /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     complement(3808..3827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4621..5209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5110..5129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcgct accggtcgcc accatggtga
     1081 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1141 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1201 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1261 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1321 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1381 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1441 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1501 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1561 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1621 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1681 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1741 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggactcg
     1801 agatgacgga atataagctg gtggtggtgg gcgccggcgg tgtgggcaag agtgcgctga
     1861 ccatccagct gatccagaac cattttgtgg acgaatacga ccccactata gaggattcct
     1921 accggaagca ggtggtcatt gatggggaga cgtgcctgtt ggacatcctg gataccgccg
     1981 gccaggagga gtacagcgcc atgcgggacc agtacatgcg caccggggag ggcttcctgt
     2041 gtgtgtttgc catcaacaac accaagtctt ttgaggacat ccaccagtac agggagcaga
     2101 tcaaacgggt gaaggactcg gatgacgtgc ccatggtgct ggtggggaac aagtgtgacc
     2161 tggctgcacg cactgtggaa tctcggcagg ctcaggacct cgcccgaagc tacggcatcc
     2221 cctacatcga gacctcggcc aagacccggc agggagtgga ggatgccttc tacacgttgg
     2281 tgcgtgagat ccggcagcac aagctgcgga agctgaaccc tcctgatgag agtggccccg
     2341 gctgcatgag ctgcaagtgt gtgctctcct gagcggccgc ttcgagcaga catgataaga
     2401 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2461 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2521 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     2581 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgggctg gcgtaatagc
     2641 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg
     2701 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2761 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2821 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2881 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2941 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3001 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3061 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3121 cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     3181 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     3241 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3301 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3361 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     3421 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     3481 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3541 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3601 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3661 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3721 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3781 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3841 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3901 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3961 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4021 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4081 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4141 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4201 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4261 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4321 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4381 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4441 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4501 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4561 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4621 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4681 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4741 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4801 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4861 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4921 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4981 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5041 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5101 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5161 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5221 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgg ctcgacagat
     5281 ct
//
