LOCUS       sequence-429696-        5653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HRAS with a G12V point mutation in human cells under a 
            tetracycline/doxycycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5653)
  AUTHORS   Zeng J, Hills SA, Ozono E, Diffley JFX
  TITLE     Cyclin E-induced replicative stress drives p53-dependent 
            whole-genome duplication.
  JOURNAL   Cell. 2023 Feb 2;186(3):528-542.e14. doi: 
            10.1016/j.cell.2022.12.036. Epub 2023 Jan 20.
  PUBMED    36681079
REFERENCE   2  (bases 1 to 5653)
  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: 
            "10.1016/j.cell.2022.12.036"; journalName: "Cell"; date: "2023-02-2-
            2"; volume: "186"; issue: "3"; pages: "528-542.e14"
COMMENT     Sequence Label: pcDNA4/TO-HRASG12V
FEATURES             Location/Qualifiers
     source          1..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1011..1037
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1047..1079,1080..1082,1083..1616)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase H-Ras"
                     /label=H-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     primer_bind     complement(1661..1678)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1667..1891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1937..2365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2024..2043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2234..2255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2374..2394)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2379..2708
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2559..2694
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2621..2640
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2756..2803
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2822..3196
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3326..3447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3363..3382)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3417..3436
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3496..3512)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3496..3512)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3509..3531)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3520..3536
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3544..3550,3551..3568,3569..3574))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3589..3610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3727..3744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3898..4486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3978..3997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4657..5448,5449..5517))
                     /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     5280..5299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5518..5622)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cgggatggag tacccatacg
     1021 atgttccaga ttacgctctc ctcctcatga cggaatataa gctggtggtg gtgggcgccg
     1081 tcggtgtggg caagagtgcg ctgaccatcc agctgatcca gaaccatttt gtggacgaat
     1141 acgaccccac tatagaggat tcctaccgga agcaggtggt cattgatggg gagacgtgcc
     1201 tgttggacat cctggatacc gccggccagg aggagtacag cgccatgcgg gaccagtaca
     1261 tgcgcaccgg ggagggcttc ctgtgtgtgt ttgccatcaa caacaccaag tcttttgagg
     1321 acatccacca gtacagggag cagatcaaac gggtgaagga ctcggatgac gtgcccatgg
     1381 tgctggtggg gaacaagtgt gacctggctg cacgcactgt ggaatctcgg caggctcagg
     1441 acctcgcccg aagctacggc atcccctaca tcgagacctc ggccaagacc cggcagggag
     1501 tggaggatgc cttctacacg ttggtgcgtg agatccggca gcacaagctg cggaagctga
     1561 accctcctga tgagagtggc cccggctgca tgagctgcaa gtgtgtgctc tcctgagcgg
     1621 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     1681 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1741 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1801 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1861 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     1921 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2341 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2401 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2461 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2521 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2581 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2641 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2701 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga
     2761 caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac
     2821 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
     2881 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
     2941 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
     3001 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
     3061 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
     3121 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
     3181 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     3241 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3301 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3361 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3421 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     3481 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3541 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3601 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3661 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3721 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3781 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3841 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3901 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3961 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4021 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4081 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4141 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4201 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4261 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4321 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4381 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4441 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4501 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4561 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4621 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4681 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4741 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4801 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4861 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4921 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4981 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5041 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5101 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5161 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5221 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5281 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5341 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5401 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5461 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5521 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5581 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5641 gccacctgac gtc
//
