LOCUS       sequence-473595-        5271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of K-RAS 4A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5271)
  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 5271)
  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 K-RAS 4A
FEATURES             Location/Qualifiers
     source          1..5271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        231..434
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     385..405
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      473..482
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(479..481,482..484,485..1195)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(479..481,482..484,485..1195)
                     /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(524..545)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(785..804)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1132..1153
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1925..2046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1962..1981)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2016..2035
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2053..2508)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2190..2211)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2402..2421
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2535..2639
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2641..2998
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2641..2832
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2664..2684)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2849..2984
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3033..3827
                     /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             3033..3827
                     /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(3087..3106)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3697..3716
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4015..4034)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4059..4106
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4435..5023
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4924..4943
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5198..5271,1..230)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
       61 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      121 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      181 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      241 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      301 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      361 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      421 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat
      481 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      541 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      601 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      661 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      721 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      781 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      841 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      901 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
      961 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1021 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1081 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1141 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     1201 actcagatct cgagctcaag cttcgatgac tgaatataaa cttgtggtag ttggagctgg
     1261 tggcgtaggc aagagtgcct tgacgataca gctaattcag aatcattttg tggacgaata
     1321 tgatccaaca atagaggatt cctacaggaa gcaagtagta attgatggag aaacctgtct
     1381 cttggatatt ctcgacacag caggtcaaga ggagtacagt gcaatgaggg accagtacat
     1441 gaggactggg gagggctttc tttgtgtatt tgccataaat aatactaaat catttgaaga
     1501 tattcaccat tatagagaac aaattaaaag agttaaggac tctgaagatg tacctatggt
     1561 cctagtagga aataaatgtg atttgccttc tagaacagta gacacaaaac aggctcagga
     1621 cttagcaaga agttatggaa ttccttttat tgaaacatca gcaaagacaa gacagagagt
     1681 ggaggatgct ttttatacat tggtgagaga gatccgacaa tacagattga aaaaaatcag
     1741 caaagaagaa aagactcctg gctgtgtgaa aattaaaaaa tgcattataa tgtaaggatc
     1801 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1861 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1921 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1981 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2041 atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2101 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2161 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2221 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2281 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2341 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2401 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2461 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2521 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2581 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2641 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2701 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2761 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2821 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2881 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2941 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3001 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3061 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3121 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3181 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3241 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3301 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3361 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3421 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3481 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3541 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3601 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3661 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3721 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3781 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3841 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3901 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3961 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4021 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4081 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4141 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4201 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4261 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4321 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4381 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4441 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4501 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4561 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4621 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4681 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4741 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4801 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4861 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4921 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4981 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5041 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5101 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
     5161 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     5221 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg a
//
