LOCUS       sequence-308700-        3949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone encoding V5-tagged mScarlet-I-fused to 
            human KRAS4A, includes a stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3949)
  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 3949)
  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 822 (pDONR221-V5-mScarlet-I-KRAS4A-WT)
FEATURES             Location/Qualifiers
     source          1..3949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..28)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(120..206)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     255..277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     269..286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     270..286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    302..401
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      420..429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             429..470
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             486..1181
                     /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(619..637)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     887..906
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1807..1906)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1923..1942)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1924..1942)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1947..1963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1947..1963)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2065..2084)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2076..2885
                     /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(2143..2162)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3032..3620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3521..3540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3774..3791
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
       61 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
      121 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
      181 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
      241 agcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
      301 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      361 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tggatccact agtgctagcg
      421 ccaccatggg taagcctatc cctaaccctc tcctcggtct cgattctacg ggtggtgctg
      481 gtggtatggt gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca
      541 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
      601 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct
      661 gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcatcaag caccccgccg
      721 acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga
      781 acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc
      841 tgatctacaa ggtgaagctc cgcggcacca acttccctcc tgacggcccc gtaatgcaga
      901 agaagacaat gggctgggaa gcatccaccg agcggttgta ccccgaggac ggcgtgctga
      961 agggcgacat taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca
     1021 agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca
     1081 agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg
     1141 agggccgcca ctccaccggc ggcatggacg agctgtacaa gtactcctcc ggactcagat
     1201 ctggaggctc cggaggcatg actgaatata aacttgtggt agttggagct ggtggcgtag
     1261 gcaagagtgc cttgacgata cagctaattc agaatcattt tgtggacgaa tatgatccaa
     1321 caatagagga ttcctacagg aagcaagtag taattgatgg agaaacctgt ctcttggata
     1381 ttctcgacac agcaggtcaa gaggagtaca gtgcaatgag ggaccagtac atgaggactg
     1441 gggagggctt tctttgtgta tttgccataa ataatactaa atcatttgaa gatattcacc
     1501 attatagaga acaaattaaa agagttaagg actctgaaga tgtacctatg gtcctagtag
     1561 gaaataaatg tgatttgcct tctagaacag tagacacaaa acaggctcag gacttagcaa
     1621 gaagttatgg aattcctttt attgaaacat cagcaaagac aagacagaga gtggaggatg
     1681 ctttttatac attggtgagg gagatccgac aatacagatt gaaaaaaatc agcaaagaag
     1741 aaaagactcc tggctgtgtg aaaattaaaa aatgcattat aatgtaaggc gcgccctcga
     1801 gagatctccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     1861 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     1921 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     1981 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     2041 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     2101 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2161 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     2221 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2281 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2341 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     2401 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2461 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2521 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2581 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     2641 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2701 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2761 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     2821 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     2881 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     2941 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     3001 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3061 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3181 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3661 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3721 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3781 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3841 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3901 acgcaattaa tacgcgtacc gctagccagg aagagtttgt agaaacgca
//
