LOCUS       sequence-301910-        5573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCS2 CMV expression vector driving V5-tagged mTagBFP2 fused human 
            KRAS4A-G12D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5573)
  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 5573)
  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 827 (pCS2-V5-mTagBFP2-hsKRAS4A-G12D)
FEATURES             Location/Qualifiers
     source          1..5573
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        74..1058
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1093..1111
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1093..1110
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1166..1190
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1209..1218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1218..1259
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1275..1985
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    complement(2611..2635)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2680..2814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2704..2723)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2758..2777
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2927..2947)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2927..2945)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2966..2982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2966..2982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2979..3001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2990..3006
                     /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(3014..3020,3021..3038,3039..3044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3059..3080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3197..3214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3368..3956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3448..3467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4127..4918,4919..4987))
                     /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     4750..4769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4988..5092)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5118..5573)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5255..5276)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5467..5486
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gtcgaccata gccaattcaa tatggcgtat atggactcat gccaattcaa
      121 tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa
      181 tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa
      241 tatggcggac ttggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt
      301 gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat
      361 ggcggccata ttggctatat gccaggatca atatataggc aatatccaat atggccctat
      421 gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc
      481 catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca
      541 tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat
      601 atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc
      661 cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga
      721 ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc
      781 cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag
      841 taacttggca agtacattac tattggaagg acgccagggt acattggcag tactcccatt
      901 gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat
      961 ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct
     1021 aatgggaggt ctatataagc aatgctcgtt tagggaaccg ccattctgcc tggggacgtc
     1081 ggagcaagct tgatttaggt gacactatag aatacaagct acttgttctt tttgcaggat
     1141 cccatcgatt cgaattcaag gatcaacaag tttgtacaaa aaagcaggct ggatccacta
     1201 gtgctagcgc caccatgggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
     1261 gtggtgctgg tggtatggtg tctaagggcg aagagctgat taaggagaac atgcacatga
     1321 agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag
     1381 gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc
     1441 ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc
     1501 acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga
     1561 gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg
     1621 acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg
     1681 tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg
     1741 gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg
     1801 caaacgccaa gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg
     1861 tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg
     1921 agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc
     1981 ttaattactc ctccggactc agatctggag gctccggagg catgactgaa tataaacttg
     2041 tggtagttgg agctgatggc gtaggcaaga gtgccttgac gatacagcta attcagaatc
     2101 attttgtgga cgaatatgat ccaacaatag aggattccta caggaagcaa gtagtaattg
     2161 atggagaaac ctgtctcttg gatattctcg acacagcagg tcaagaggag tacagtgcaa
     2221 tgagggacca gtacatgagg actggggagg gctttctttg tgtatttgcc ataaataata
     2281 ctaaatcatt tgaagatatt caccattata gagaacaaat taaaagagtt aaggactctg
     2341 aagatgtacc tatggtccta gtaggaaata aatgtgattt gccttctaga acagtagaca
     2401 caaaacaggc tcaggactta gcaagaagtt atggaattcc ttttattgaa acatcagcaa
     2461 agacaagaca gagagtggag gatgcttttt atacattggt gagggagatc cgacaataca
     2521 gattgaaaaa aatcagcaaa gaagaaaaga ctcctggctg tgtgaaaatt aaaaaatgca
     2581 ttataatgta aggcgcgccc tcgagagatc tcccagcttt cttgtacaaa gtggttgatc
     2641 ctctcgagcc tctagaacta tagtgagtcg tattacgtag atccagacat gataagatac
     2701 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     2761 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     2821 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc
     2881 gcggccgcgg cgccaatgca ttgggcccgg tacccagctt ttgttccctt tagtgagggt
     2941 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3001 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3061 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3121 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3181 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3241 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3301 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3361 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3421 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3481 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3541 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3601 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3661 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3721 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3781 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     3841 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3901 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3961 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4021 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4081 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4141 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4201 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4261 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4321 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4381 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4441 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4501 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4561 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4621 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4681 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4741 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4801 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4861 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4921 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4981 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5041 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5101 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5161 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5221 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5281 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5341 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5401 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5461 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5521 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgt
//
