LOCUS       sequence-486840-        4243 bp    DNA     circular SYN 22-SEP-2025
DEFINITION  Gateway 3' entry clone containing mCherry followed by a T2A cleavage
            peptide and then human HRAS G12V. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4243)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, Mosser LE, Fujimoto E, Chien CB, Kwan KM, 
            Arenkiel BR, Devine WP, Wythe JD
  TITLE     Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 4243)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: p3E mCherry-T2A-hsHRAS-G12V-pA (JDW 1188)
FEATURES             Location/Qualifiers
     source          1..4243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      772..781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1191..1210
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1385..1408
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1504..1557
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1564..1596,1597..1599,1600..2133)
                     /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"
     polyA_signal    2191..2325
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2215..2234)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2269..2288
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2352..2447)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2484..2503)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2485..2503)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2508..2524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2508..2524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2626..2645)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2637..3446
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2704..2723)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3593..4181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4082..4101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggcgg atccgatatc gtcgactcga ggccaccatg
      781 gtgagcaagg gcgaggagga caacatggcc atcatcaagg agttcatgcg cttcaaggtg
      841 cacatggagg gctccgtgaa cggccacgag ttcgagatag aaggagaagg agaaggacgg
      901 ccatatgaag gtacgcaaac agctaaactc aaggtgacca agggcggccc cctgcccttc
      961 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1021 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1081 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1141 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1201 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1261 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgccgag
     1321 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1381 atcaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc
     1441 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagggtac caagggctcc
     1501 ggcgagggca ggggaagtct actaacatgc ggggacgtgg aggaaaatcc cggcccagaa
     1561 ttcatgacag aatacaagct ggtggtggtg ggcgccgtcg gtgtgggcaa gagtgcgctg
     1621 accatccagc tgatccagaa ccattttgtg gacgaatacg accccactat agaggattcc
     1681 taccggaagc aggtggtcat tgatggggag acgtgcctgt tggacatcct ggataccgcc
     1741 ggccaggagg agtacagcgc catgcgggac cagtacatgc gcaccgggga gggcttcctg
     1801 tgtgtgtttg ccatcaacaa caccaagtct tttgaggaca tccaccagta cagggagcag
     1861 atcaaacggg tgaaggactc ggatgacgtg cccatggtgc tggtggggaa caagtgtgac
     1921 ctggctgcac gcactgtgga atctcggcag gctcaggacc tcgcccgaag ctacggcatc
     1981 ccctacatcg agacctcggc caagacccgg cagggagtgg aggatgcctt ctacacgttg
     2041 gtgcgtgaga tccggcagca caagctgcgg aagctgaacc ctcctgatga gagtggcccc
     2101 ggctgcatga gctgcaagtg tgtgctctcc tgagcggccg caggcgcgcc acgcgtaagc
     2161 ttctgcagtc tagacgtata cgtagttaac gatccagaca tgataagata cattgatgag
     2221 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2281 gctattgctt tatttgtaac cattataagc tgcaataaac aagtttacgt aaacactagt
     2341 aacaacaatt gcaactttat tatacaaagt tggcattata aaaaagcatt gcttatcaat
     2401 ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgga gctccatggt
     2461 agcgttaacg cggccgcgat atcccctata gtgagtcgta ttacatggtc atagctgttt
     2521 cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2581 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     2641 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     2701 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     2761 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     2821 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     2881 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     2941 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     3001 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     3061 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     3121 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     3181 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     3241 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     3301 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     3361 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     3421 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     3481 gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt
     3541 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3601 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3661 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3721 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3781 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3841 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3901 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3961 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4021 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4081 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4141 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4201 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
