LOCUS       sequence-473602-        5287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of N-RAS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5287)
  AUTHORS   Kuchay S, Wang H, Marzio A, Jain K, Homer H, Fehrenbacher N, Philips
            MR, Zheng N, Pagano M
  TITLE     GGTase3 is a newly identified geranylgeranyltransferase targeting a 
            ubiquitin ligase.
  JOURNAL   Nat Struct Mol Biol. 2019 Jul;26(7):628-636. doi: 
            10.1038/s41594-019-0249-3. Epub 2019 Jun 17.
  PUBMED    31209342
REFERENCE   2  (bases 1 to 5287)
  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/s41594-019-0249-3"; journalName: "Nat Struct Mol Biol"; 
            date: "2019-07"; volume: "26"; issue: "7"; pages: "628-636"
COMMENT     Sequence Label: pEGFPc3 N-RAS
FEATURES             Location/Qualifiers
     source          1..5287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..152
                     /gene="bla"
                     /label=AmpR promoter
     promoter        154..511
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        154..345
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(177..197)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      362..497
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             546..1340
                     /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             546..1340
                     /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(600..619)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1210..1229
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1528..1547)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1572..1619
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1948..2536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2437..2456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2711..3014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3015..3218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3169..3189
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3257..3266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3263..3265,3266..3268,3269..3979)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3263..3265,3266..3268,3269..3979)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3308..3329)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3569..3588)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3916..3937
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4725..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4762..4781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4816..4835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(join(4853..5287,1..21))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4990..5011)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5202..5221
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
       61 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      121 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
      181 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      241 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
      301 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
      361 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
      421 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
      481 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
      541 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
      601 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
      661 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
      721 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
      781 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
      841 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
      901 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
      961 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1021 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     1081 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     1141 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     1201 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     1261 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     1321 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     1381 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     1441 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     1501 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     1561 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     1621 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     1681 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     1741 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     1801 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1861 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1921 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1981 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2041 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2101 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2161 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2221 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2281 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2341 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2401 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2461 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2521 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2581 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2641 cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     2701 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2761 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2821 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2881 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2941 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     3001 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     3061 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     3121 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     3181 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
     3241 gctagcgcta ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     3301 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     3361 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3421 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     3481 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3541 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3601 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3661 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3721 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3781 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3841 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3901 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3961 catggacgag ctgtacaagt actcagatct cgagctcaag cttcgaattc tgatgactga
     4021 gtataaactg gtggtggttg gagcaggtgg tgttgggaaa agcgcactga caatccagct
     4081 aatccagaac cactttgtag atgaatatga tcccaccata gaggattctt acagaaaaca
     4141 agtggttata gatggtgaaa cctgtttgtt ggacatactg gatacagctg gacaagaaga
     4201 gtacagtgcc atgagagacc aatacatgag gacaggcgaa ggcttcctct gtgtatttgc
     4261 catcaataat agcaagtcat ttgcggatat taacctctac agggagcaga ttaagcgagt
     4321 aaaagactcg gatgatgtac ctatggtgct agtgggaaac aagtgtgatt tgccaacaag
     4381 gacagttgat acaaaacaag cccacgaact ggccaagagt tacgggattc cattcattga
     4441 aacctcagcc aagaccagac agggtgttga agatgctttt tacacactgg taagagaaat
     4501 acgccagtac cgaatgaaaa aactcaacag cagtgatgat gggactcagg gttgtatggg
     4561 attgccatgt gtggtgatgt aatctagagg gcccgggatc caccggatct agataactga
     4621 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     4681 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     4741 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4801 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     4861 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     4921 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     4981 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5041 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5101 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5161 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5221 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5281 gcgctta
//
