LOCUS       sequence-308697-        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"
     terminator      complement(92..178)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     227..249
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     241..258
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     242..258
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    274..373
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      386..395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             395..436
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             452..1147
                     /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(585..603)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     853..872
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1773..1872)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1889..1908)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1890..1908)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1913..1929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1913..1929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2031..2050)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2042..2851
                     /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(2109..2128)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2998..3586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3487..3506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3740..3757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3916..3943)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
       61 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
      121 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
      181 gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt
      241 tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata
      301 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac
      361 aaaaaagcag gctggatcca ctagcgccac catgggtaag cctatcccta accctctcct
      421 cggtctcgat tctacgggtg gtgctggtgg tatggtgagc aagggcgagg cagtgatcaa
      481 ggagttcatg cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat
      541 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
      601 caagggtggc cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc
      661 cagggccttc atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga
      721 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca
      781 ggacacctcc ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt
      841 ccctcctgac ggccccgtaa tgcagaagaa gacaatgggc tgggaagcat ccaccgagcg
      901 gttgtacccc gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga
      961 cggcggccgc tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat
     1021 gcccggcgcc tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac
     1081 cgtggtggaa cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct
     1141 gtacaagtac tcctccggac tcagatctgg aggctccgga ggcatgactg aatataaact
     1201 tgtggtagtt ggagctgttg gcgtaggcaa gagtgccttg acgatacagc taattcagaa
     1261 tcattttgtg gacgaatatg atccaacaat agaggattcc tacaggaagc aagtagtaat
     1321 tgatggagaa acctgtctct tggatattct cgacacagca ggtcaagagg agtacagtgc
     1381 aatgagggac cagtacatga ggactgggga gggctttctt tgtgtatttg ccataaataa
     1441 tactaaatca tttgaagata ttcaccatta tagagaacaa attaaaagag ttaaggactc
     1501 tgaagatgta cctatggtcc tagtaggaaa taaatgtgat ttgccttcta gaacagtaga
     1561 cacaaaacag gctcaggact tagcaagaag ttatggaatt ccttttattg aaacatcagc
     1621 aaagacaaga cagagagtgg aggatgcttt ttatacattg gtgagggaga tccgacaata
     1681 cagattgaaa aaaatcagca aagaagaaaa gactcctggc tgtgtgaaaa ttaaaaaatg
     1741 cattataatg taaggcgcgc cctcgagaga tctcccagct ttcttgtaca aagttggcat
     1801 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     1861 aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
     1921 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1981 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     2041 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     2101 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     2161 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     2221 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     2281 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2341 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2401 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2461 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2521 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2581 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2641 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2701 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2761 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2821 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     2881 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     2941 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3001 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3061 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3121 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3181 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3241 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3301 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3361 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3421 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3481 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3541 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3601 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3661 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3721 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     3781 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     3841 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga
     3901 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tct
//
