LOCUS       sequence-473616-        5269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of K-RAS 4B in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5269)
  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 5269)
  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 K-RAS 4B
FEATURES             Location/Qualifiers
     source          1..5269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..143
                     /gene="bla"
                     /label=AmpR promoter
     promoter        145..502
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        145..336
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(168..188)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      353..488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             537..1331
                     /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             537..1331
                     /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(591..610)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1201..1220
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1519..1538)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1563..1610
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1939..2527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2428..2447
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2702..3005
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3006..3209
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3160..3180
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3248..3257
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3254..3256,3257..3259,3260..3970)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3254..3256,3257..3259,3260..3970)
                     /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(3299..3320)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3560..3579)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3907..3928
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3995..4561
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    4698..4819
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4735..4754)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4789..4808
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(join(4826..5269,1..12))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4963..4984)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5175..5194
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
       61 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
      121 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
      181 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
      241 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      301 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
      361 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
      421 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
      481 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
      541 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
      601 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
      661 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
      721 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
      781 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
      841 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
      901 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
      961 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     1021 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     1081 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     1141 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     1201 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     1261 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     1321 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     1381 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     1441 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     1501 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     1561 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     1621 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     1681 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     1741 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     1801 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1861 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1921 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1981 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2041 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2101 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2161 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2221 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2281 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2341 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2401 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2461 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2521 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2581 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     2641 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2701 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2761 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2821 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2881 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2941 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     3001 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     3061 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     3121 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     3181 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct
     3241 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3301 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3361 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3421 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     3481 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3541 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3601 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3661 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     3721 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     3781 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3841 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3901 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     3961 gctgtacaag tactcagatc tcgagctcaa gcttatgact gaatataaac ttgtggtagt
     4021 tggagctggt ggcgtaggca agagtgcctt gacgatacag ctaattcaga atcattttgt
     4081 ggacgaatat gatccaacaa tagaggattc ctacaggaag caagtagtaa ttgatggaga
     4141 aacctgtctc ttggatattc tcgacacagc aggtcaagag gagtacagtg caatgaggga
     4201 ccagtacatg aggactgggg agggctttct ttgtgtattt gccataaata atactaaatc
     4261 atttgaagat attcaccatt atagagaaca aattaaaaga gttaaggact ctgaagatgt
     4321 acctatggtc ctagtaggaa ataaatgtga tttgccttcc agaacagtag acacaaaaca
     4381 ggctcaggac ttagcaagaa gttatggaat tccttttatt gaaacatcag caaagacaag
     4441 acagggtgtt gatgatgcct tctatacatt agttcgagaa attcgaaaac ataaagaaaa
     4501 gatgagcaaa gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg taattatgta
     4561 agggcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     4621 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     4681 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4741 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4801 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4861 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4921 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4981 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5041 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5101 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5161 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5221 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgc
//
