LOCUS       sequence-301907-        3943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone encoding V5-tagged mScarlet-I-fused to 
            human KRAS4A with G12V mutation, includes a stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3943)
  AUTHORS   Fish JE, Flores-Suarez CP, Boudreau E, Herman AM, Gutierrez MC, 
            Gustafson D, DiStefano PV, Cui M, Chen Z, Berman De Ruiz K, 
            Schexnayder TS, Ward CS, Radovanovic I, Wythe JD
  TITLE     Somatic Gain of KRAS Function in the Endothelium is Sufficient to 
            Cause Vascular Malformations that Require MEK but not PI3K 
            Signaling.
  JOURNAL   Circ Res. 2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500.
  PUBMED    32552404
REFERENCE   2  (bases 1 to 3943)
  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: "Circ Res. 
            2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500."
COMMENT     Sequence Label: JDW 823 (pDONR221-V5-mScarlet-I-hsKRAS4A-G12V)
FEATURES             Location/Qualifiers
     source          1..3943
                     /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    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      681..690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             690..731
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             747..1442
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(880..898)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1148..1167
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2068..2167)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2184..2203)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2185..2203)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2208..2224)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2208..2224)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2326..2345)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2337..3146
                     /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(2404..2423)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3293..3881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3782..3801
                     /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 tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgg atccactagc gccaccatgg gtaagcctat ccctaaccct ctcctcggtc
      721 tcgattctac gggtggtgct ggtggtatgg tgagcaaggg cgaggcagtg atcaaggagt
      781 tcatgcggtt caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg
      841 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
      901 gtggccccct gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg
      961 ccttcatcaa gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct
     1021 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca
     1081 cctccctgga ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc
     1141 ctgacggccc cgtaatgcag aagaagacaa tgggctggga agcatccacc gagcggttgt
     1201 accccgagga cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg
     1261 gccgctacct ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg
     1321 gcgcctacaa cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg
     1381 tggaacagta cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     1441 agtactcctc cggactcaga tctggaggct ccggaggcat gactgaatat aaacttgtgg
     1501 tagttggagc tgttggcgta ggcaagagtg ccttgacgat acagctaatt cagaatcatt
     1561 ttgtggacga atatgatcca acaatagagg attcctacag gaagcaagta gtaattgatg
     1621 gagaaacctg tctcttggat attctcgaca cagcaggtca agaggagtac agtgcaatga
     1681 gggaccagta catgaggact ggggagggct ttctttgtgt atttgccata aataatacta
     1741 aatcatttga agatattcac cattatagag aacaaattaa aagagttaag gactctgaag
     1801 atgtacctat ggtcctagta ggaaataaat gtgatttgcc ttctagaaca gtagacacaa
     1861 aacaggctca ggacttagca agaagttatg gaattccttt tattgaaaca tcagcaaaga
     1921 caagacagag agtggaggat gctttttata cattggtgag ggagatccga caatacagat
     1981 tgaaaaaaat cagcaaagaa gaaaagactc ctggctgtgt gaaaattaaa aaatgcatta
     2041 taatgtaagg cgcgccctcg agagatctcc cagctttctt gtacaaagtt ggcattataa
     2101 gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca
     2161 ttatttgcca tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt
     2221 cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2281 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     2341 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     2401 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     2461 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     2521 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     2581 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     2641 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     2701 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     2761 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     2821 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     2881 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     2941 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     3001 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     3061 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     3121 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     3181 gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt
     3241 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3301 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3361 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3421 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3481 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3541 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3601 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3661 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3721 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3781 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3841 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3901 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
