LOCUS       sequence-308705-        5549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCS2 CMV expression vector driving V5-tagged mScarlet-I-fused human 
            KRAS4A-G12V.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5549)
  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 5549)
  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 837 (pCS2-V5-mScarlet-I-hsKRAS4A-G12V)
FEATURES             Location/Qualifiers
     source          1..5549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      145..154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             154..195
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             211..906
                     /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(344..362)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     612..631
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1532..1556)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1601..1735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1625..1644)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1679..1698
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1848..1868)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1848..1866)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1887..1903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1887..1903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1900..1922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1911..1927
                     /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(1935..1941,1942..1959,1960..1965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1980..2001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2118..2135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2289..2877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2369..2388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3048..3839,3840..3908))
                     /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     3671..3690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3909..4013)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4039..4494)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4176..4197)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4388..4407
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4565..5549
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctggatccac tagcgccacc atgggtaagc ctatccctaa ccctctcctc
      181 ggtctcgatt ctacgggtgg tgctggtggt atggtgagca agggcgaggc agtgatcaag
      241 gagttcatgc ggttcaaggt gcacatggag ggctccatga acggccacga gttcgagatc
      301 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
      361 aagggtggcc ccctgccctt ctcctgggac atcctgtccc ctcagttcat gtacggctcc
      421 agggccttca tcaagcaccc cgccgacatc cccgactact ataagcagtc cttccccgag
      481 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac cgtgacccag
      541 gacacctccc tggaggacgg caccctgatc tacaaggtga agctccgcgg caccaacttc
      601 cctcctgacg gccccgtaat gcagaagaag acaatgggct gggaagcatc caccgagcgg
      661 ttgtaccccg aggacggcgt gctgaagggc gacattaaga tggccctgcg cctgaaggac
      721 ggcggccgct acctggcgga cttcaagacc acctacaagg ccaagaagcc cgtgcagatg
      781 cccggcgcct acaacgtcga ccgcaagttg gacatcacct cccacaacga ggactacacc
      841 gtggtggaac agtacgaacg ctccgagggc cgccactcca ccggcggcat ggacgagctg
      901 tacaagtact cctccggact cagatctgga ggctccggag gcatgactga atataaactt
      961 gtggtagttg gagctgttgg cgtaggcaag agtgccttga cgatacagct aattcagaat
     1021 cattttgtgg acgaatatga tccaacaata gaggattcct acaggaagca agtagtaatt
     1081 gatggagaaa cctgtctctt ggatattctc gacacagcag gtcaagagga gtacagtgca
     1141 atgagggacc agtacatgag gactggggag ggctttcttt gtgtatttgc cataaataat
     1201 actaaatcat ttgaagatat tcaccattat agagaacaaa ttaaaagagt taaggactct
     1261 gaagatgtac ctatggtcct agtaggaaat aaatgtgatt tgccttctag aacagtagac
     1321 acaaaacagg ctcaggactt agcaagaagt tatggaattc cttttattga aacatcagca
     1381 aagacaagac agagagtgga ggatgctttt tatacattgg tgagggagat ccgacaatac
     1441 agattgaaaa aaatcagcaa agaagaaaag actcctggct gtgtgaaaat taaaaaatgc
     1501 attataatgt aaggcgcgcc ctcgagagat ctcccagctt tcttgtacaa agtggttgat
     1561 cctctcgagc ctctagaact atagtgagtc gtattacgta gatccagaca tgataagata
     1621 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1681 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     1741 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt
     1801 cgcggccgcg gcgccaatgc attgggcccg gtacccagct tttgttccct ttagtgaggg
     1861 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1921 ctcacaattc cacacaacat acgagccggg agcataaagt gtaaagcctg gggtgcctaa
     1981 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2041 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2101 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2161 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2221 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2281 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2341 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2401 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2461 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2521 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2581 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2641 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2701 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2761 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2821 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2881 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2941 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3001 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3061 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3121 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3181 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3241 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3301 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3361 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3421 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3481 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3541 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3601 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3661 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3721 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3781 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3841 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3901 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3961 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4021 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     4081 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     4141 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     4201 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     4261 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     4321 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4381 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4441 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     4501 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     4561 gccagtcgac cgccaattca atatggcgta tatggactca tgccaattca atatggtgga
     4621 tctggacctg tgccaattca atatggcgta tatggactcg tgccaattca atatggtgga
     4681 tctggacccc agccaattca atatggcgga cttggcacca tgccaattca atatggcgga
     4741 cctggcactg tgccaactgg ggaggggtct acttggcacg gtgccaagtt tgaggagggg
     4801 tcttggccct gtgccaagtc cgccatattg aattggcatg gtgccaataa tggcggccat
     4861 attggctata tgccaggatc aatatatagg caatatccaa tatggcccta tgccaatatg
     4921 gctattggcc aggttcaata ctatgtattg gccctatgcc atatagtatt ccatatatgg
     4981 gttttcctat tgacgtagat agcccctccc aatgggcggt cccatatacc atatatgggg
     5041 cttcctaata ccgcccatag ccactccccc attgacgtca atggtctcta tatatggtct
     5101 ttcctattga cgtcatatgg gcggtcctat tgacgtatat ggcgcctccc ccattgacgt
     5161 caattacggt aaatggcccg cctggctcaa tgcccattga cgtcaatagg accacccacc
     5221 attgacgtca atgggatggc tcattgccca ttcatatccg ttctcacgcc ccctattgac
     5281 gtcaatgacg gtaaatggcc cacttggcag tacatcaata tctattaata gtaacttggc
     5341 aagtacatta ctattggaag tacgccaggg tacattggca gtactcccat tgacgtcaat
     5401 ggcggtaaat ggcccgcgat ggctgccaag tacatcccca ttgacgtcaa tggggagggg
     5461 caatgacgca aatgggcgtt ccattgacgt aaatgggcgg taggcgtgcc taatgggagg
     5521 tctatataag caatgctcgt ttagggaac
//
