LOCUS       sequence-473594-        5287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of H-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: pEGFPc1 H-RAS
FEATURES             Location/Qualifiers
     source          1..5287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        258..461
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     412..432
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      500..509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(506..508,509..511,512..1222)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(506..508,509..511,512..1222)
                     /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(551..572)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(812..831)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1159..1180
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1262..1831
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    1968..2089
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2005..2024)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2059..2078
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2096..2551)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2233..2254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2445..2464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2578..2682
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2684..3041
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2684..2875
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2707..2727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2892..3027
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3076..3870
                     /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             3076..3870
                     /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(3130..3149)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3740..3759
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4058..4077)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4102..4149
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4478..5066
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4967..4986
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5241..5287,1..257)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
       61 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      121 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      181 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      241 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      301 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      361 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      421 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      481 tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
      541 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      601 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      661 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      721 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      781 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      841 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      901 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
      961 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1021 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1081 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1141 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1201 cggcatggac gagctgtaca agtccggact cagatctcga gctcaagctt cgaattctgc
     1261 aatgacggaa tataagcttg ttgttgttgg cgccggcggt gtgggcaaga gtgcgctgac
     1321 catccagctg atccagaacc attttgtgga cgaatacgac cccactatag aggattccta
     1381 ccggaagcag gtggtcattg atggggagac gtgcctgttg gacatcctgg ataccgccgg
     1441 ccaggaggag tacagcgcca tgcgggacca gtacatgcgc accggggagg gcttcctgtg
     1501 tgtgtttgcc atcaacaaca ccaagtcttt tgaggacatc caccagtaca gggagcagat
     1561 caaacgggtg aaggactcgg atgacgtgcc catggtgctg gtggggaaca agtgtgacct
     1621 ggctgcacgc actgtggaat ctcggcaggc tcaggacctc gcccgaagct acggcatccc
     1681 ctacatcgag acctcggcca agacccggca gggagtggag gatgccttct acacgttggt
     1741 gcgtgagatc cggcagcaca agctgcggaa gctgaaccct cctgatgaga gtggccccgg
     1801 ctgcatgagc tgcaagtgtg tgctctcctg agggcccggg atccaccgga tctagataac
     1861 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1921 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1981 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2041 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt
     2101 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2161 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2221 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2281 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2341 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2401 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2461 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2521 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2581 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2641 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2701 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2761 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2821 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2881 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2941 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3001 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     3061 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3121 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3181 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3241 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3301 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3361 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3421 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3481 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3541 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3601 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3661 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3721 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3781 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3841 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3901 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3961 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4021 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     4081 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     4141 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     4201 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     4261 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     4321 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4381 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4441 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4501 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4561 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4621 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4681 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4741 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4801 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4861 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4921 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4981 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5041 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5101 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5161 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
     5221 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     5281 cccaacg
//
