LOCUS       sequence-8809-ex        5311 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 5311)
  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 5311)
  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 S17N
FEATURES             Location/Qualifiers
     source          1..5311
                     /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      1097..1106
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1103..1105,1106..1108,1109..1819)
                     /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(1148..1169)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1409..1428)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1756..1777
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1832..2401
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKNALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    2425..2546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2436..2455)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2490..2509
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2727..3182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2814..2833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3024..3045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3249..3268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3368..3390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3428..3446)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3514..3618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3619..3687,3688..4479)
                     /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(3837..3856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4650..5238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5139..5158
                     /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 ggctagcctc gagaattcac gcgtggtacc
     1081 tctagcgcta ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     1141 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1201 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1261 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1321 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1381 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1441 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1501 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1561 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1621 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1681 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     1741 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1801 catggacgag ctgtacaagt ccggactcga gatgacggaa tataagctgg tggtggtggg
     1861 cgccggcggt gtgggcaaga atgcgctgac catccagctg atccagaacc attttgtgga
     1921 cgaatacgac cccactatag aggattccta ccggaagcag gtggtcattg atggggagac
     1981 gtgcctgttg gacatcctgg ataccgccgg ccaggaggag tacagcgcca tgcgggacca
     2041 gtacatgcgc accggggagg gcttcctgtg tgtgtttgcc atcaacaaca ccaagtcttt
     2101 tgaggacatc caccagtaca gggagcagat caaacgggtg aaggactcgg atgacgtgcc
     2161 catggtgctg gtggggaaca agtgtgacct ggctgcacgc actgtggaat ctcggcaggc
     2221 tcaggacctc gcccgaagct acggcatccc ctacatcgag acctcggcca agacccggca
     2281 gggagtggag gatgccttct acacgttggt gcgtgagatc cggcagcaca agctgcggaa
     2341 gctgaaccct cctgatgaga gtggccccgg ctgcatgagc tgcaagtgtg tgctctcctg
     2401 agcggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     2461 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2521 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     2581 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     2641 tcgataagga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2701 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2761 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2821 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2881 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2941 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3001 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3061 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3121 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3181 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     3241 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     3301 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3361 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     3421 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3481 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3541 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3601 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3661 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3721 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3781 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3841 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3901 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3961 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4021 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4081 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4141 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4201 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4261 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4321 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4381 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4441 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4501 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4561 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4621 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4681 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4741 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4801 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4861 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4921 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4981 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5041 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5101 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5161 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5221 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5281 tggccttttg ctcacatggc tcgacagatc t
//
