LOCUS       sequence-308703-        5461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCS2 CMV expression vector driving EYFP fused human KRAS4B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5461)
  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 5461)
  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 763 (pCS2-EYFP-hsKRAS4B-WT)
FEATURES             Location/Qualifiers
     source          1..5461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        396..414
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     396..413
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    469..493
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      496..505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(502..504,505..507,508..1218)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(547..568)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(808..827)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1155..1176
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1237..1803
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1805..1829)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1874..2008
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1898..1917)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1952..1971
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2121..2141)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2121..2139)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2160..2176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2160..2176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2173..2195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2184..2200
                     /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(2208..2214,2215..2232,2233..2238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2253..2274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2391..2408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2562..3150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2642..2661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3321..4112,4113..4181))
                     /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     3944..3963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4182..4286)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4312..4767)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4470)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4661..4680
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4838..5461,1..361)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 aatgcccatt gacgtcaata ggaccaccca ccattgacgt caatgggatg gctcattgcc
       61 cattcatatc cgttctcacg ccccctattg acgtcaatga cggtaaatgg cccacttggc
      121 agtacatcaa tatctattaa tagtaacttg gcaagtacat tactattgga agtacgccag
      181 ggtacattgg cagtactccc attgacgtca atggcggtaa atggcccgcg atggctgcca
      241 agtacatccc cattgacgtc aatggggagg ggcaatgacg caaatgggcg ttccattgac
      301 gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc gtttagggaa
      361 ccgccattct gcctggggac gtcggagcaa gcttgattta ggtgacacta tagaatacaa
      421 gctacttgtt ctttttgcag gatcccatcg attcgaattc aaggatcaac aagtttgtac
      481 aaaaaagcag gctaagccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      541 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      601 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      661 ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc
      721 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      781 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      841 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      901 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
      961 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1021 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1081 gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac
     1141 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1201 atggacgagc tgtacaagtc cggactcaga tccaccatga ctgaatataa acttgtggta
     1261 gttggagctg gtggcgtagg caagagtgcc ttgacgatac agctaattca gaatcatttt
     1321 gtggacgaat atgatccaac aatagaggat tcctacagga agcaagtagt aattgatgga
     1381 gaaacctgtc tcttggatat tctcgacaca gcaggtcaag aggagtacag tgcaatgagg
     1441 gaccagtaca tgaggactgg ggagggcttt ctttgtgtat ttgccataaa taatactaaa
     1501 tcatttgaag atattcacca ttatagagaa caaattaaaa gagttaagga ctctgaagat
     1561 gtacctatgg tcctagtagg aaataaatgt gatttgcctt ccagaacagt agacacaaaa
     1621 caggctcagg acttagcaag aagttatgga attcctttta ttgaaacatc agcaaagaca
     1681 agacagggtg ttgatgatgc cttctataca ttagttcgag aaattcgaaa acataaagaa
     1741 aagatgagca aagatggtaa aaagaagaaa aagaagtcaa agacaaagtg tgtaattatg
     1801 taagacccag ctttcttgta caaagtggtt gatcctctcg agcctctaga actatagtga
     1861 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
     1921 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     1981 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     2041 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc
     2101 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     2161 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2221 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2281 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2341 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2401 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2461 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2521 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2581 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2641 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2701 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2761 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2821 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2881 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2941 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3001 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3061 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3121 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3181 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3241 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3301 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3361 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3421 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3481 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3541 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3601 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3661 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3721 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3781 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3841 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3901 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3961 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4021 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4081 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4141 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4201 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4261 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     4321 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4381 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4441 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4501 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4561 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4621 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4681 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4741 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     4801 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     4861 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     4921 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     4981 ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg
     5041 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     5101 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     5161 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     5221 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     5281 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     5341 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     5401 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     5461 c
//
