LOCUS       sequence-275165-        4414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR SONIC:  HRAS G12V donor plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4414)
  AUTHORS   Mou H, Ozata DM, Smith JL, Sheel A, Kwan SY, Hough S, Kucukural A, 
            Kennedy Z, Cao Y, Xue W
  TITLE     CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in 
            vivo cancer modeling.
  JOURNAL   Genome Med. 2019 Apr 16;11(1):21. doi: 10.1186/s13073-019-0627-9.
  PUBMED    30987660
REFERENCE   2  (bases 1 to 4414)
  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"; journalName: "Genome 
            Med."; date: "2019-04-16"; volume: "11(1):21. doi"; pages: " 
            10.1186/s13073-019-0627-9"
COMMENT     Sequence Label: H517 SONIC HRASV12 donor
FEATURES             Location/Qualifiers
     source          1..4414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..568
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    18..570
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(162..179)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     389..408
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(582..614,615..617,618..1151)
                     /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(1183..1200)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1189..1413
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(1414..1447)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1493..1511)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1532..1548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1532..1548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1545..1567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1556..1572
                     /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(1580..1586,1587..1604,1605..1610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1625..1646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1763..1780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1934..2522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2014..2033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2693..3484,3485..3553))
                     /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     3316..3335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3554..3658)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3684..4139)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3821..3842)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4033..4052
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4266..4288
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4280..4297
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4281..4297
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4304..4323
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4304..4322
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4355..4371
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4355..4371
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
       61 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
      121 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
      181 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
      241 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
      301 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
      361 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
      421 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtacacatg
      481 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
      541 ggttttcctt tgaaaaacac gatgataata tggccaccac catgacggaa tataagctgg
      601 tggtggtggg cgccgtcggt gtgggcaaga gtgcgctgac catccagctg atccagaacc
      661 attttgtgga cgaatacgac cccactatag aggattccta ccggaagcag gtggtcattg
      721 atggggagac gtgcctgttg gacatcctgg ataccgccgg ccaggaggag tacagcgcca
      781 tgcgggacca gtacatgcgc accggggagg gcttcctgtg tgtgtttgcc atcaacaaca
      841 ccaagtcttt tgaggacatc caccagtaca gggagcagat caaacgggtg aaggactcgg
      901 atgacgtgcc catggtgctg gtggggaaca agtgtgacct ggctgcacgc actgtggaat
      961 ctcggcaggc tcaggacctc gcccgaagct acggcatccc ctacatcgag acctcggcca
     1021 agacccggca gggagtggag gatgccttct acacgttggt gcgtgagatc cggcagcaca
     1081 agctgcggaa gctgaaccct cctgatgaga gtggccccgg ctgcatgagc tgcaagtgtg
     1141 tgctctcctg aatgcatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta
     1201 gttgccagct atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1261 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1321 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1381 gcaggcatgc tggggatgcg gtgggctcta tggataactt cgtatagcat acattatacg
     1441 aagttatcgg aacccttaat gtcgaggggg ggcccggtac cagcttttgt tccctttagt
     1501 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1561 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1621 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1741 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3661 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     3721 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3781 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3841 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3901 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3961 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4021 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4081 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4141 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4201 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     4261 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
     4321 gggcgaattg gagctccacc gcggtggcgg ccgctctaga actagtggat ccgagatcga
     4381 gtgccgcatc accggcaatt gacgcgtgct cctc
//
