LOCUS       sequence-308702-        5570 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 5570)
  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 5570)
  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..5570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        721..739
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     721..738
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    794..818
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      837..846
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             846..887
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             903..1613
                     /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(2239..2263)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2308..2442
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2332..2351)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2386..2405
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2555..2575)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2555..2573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2594..2610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2594..2610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2607..2629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2618..2634
                     /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(2642..2648,2649..2666,2667..2672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2687..2708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2825..2842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2996..3584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3076..3095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3755..4546,4547..4615))
                     /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     4378..4397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4616..4720)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4746..5201)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4883..4904)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5095..5114
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5272..5570,1..686)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ggctatatgc caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct
       61 attggccagg ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt
      121 ttcctattga cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt
      181 cctaataccg cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc
      241 ctattgacgt catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa
      301 ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt
      361 gacgtcaatg ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc
      421 aatgacggta aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag
      481 tacattacta ttggaagtac gccagggtac attggcagta ctcccattga cgtcaatggc
      541 ggtaaatggc ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa
      601 tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct
      661 atataagcaa tgctcgttta gggaaccgcc attctgcctg gggacgtcgg agcaagcttg
      721 atttaggtga cactatagaa tacaagctac ttgttctttt tgcaggatcc catcgattcg
      781 aattcaagga tcaacaagtt tgtacaaaaa agcaggctgg atccactagt gctagcgcca
      841 ccatgggtaa gcctatccct aaccctctcc tcggtctcga ttctacgggt ggtgctggtg
      901 gtatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
      961 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     1021 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     1081 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     1141 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     1201 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     1261 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     1321 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     1381 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     1441 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     1501 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     1561 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aattactcct
     1621 ccggactcag atctggaggc tccggaggca tgactgaata taaacttgtg gtagttggag
     1681 ctgatggcgt aggcaagagt gccttgacga tacagctaat tcagaatcat tttgtggacg
     1741 aatatgatcc aacaatagag gattcctaca ggaagcaagt agtaattgat ggagaaacct
     1801 gtctcttgga tattctcgac acagcaggtc aagaggagta cagtgcaatg agggaccagt
     1861 acatgaggac tggggagggc tttctttgtg tatttgccat aaataatact aaatcatttg
     1921 aagatattca ccattataga gaacaaatta aaagagttaa ggactctgaa gatgtaccta
     1981 tggtcctagt aggaaataaa tgtgatttgc cttctagaac agtagacaca aaacaggctc
     2041 aggacttagc aagaagttat ggaattcctt ttattgaaac atcagcaaag acaagacaga
     2101 gagtggagga tgctttttat acattggtga gggagatccg acaatacaga ttgaaaaaaa
     2161 tcagcaaaga agaaaagact cctggctgtg tgaaaattaa aaaatgcatt ataatgtaag
     2221 gcgcgccctc gagagatctc ccagctttct tgtacaaagt ggttgatcct ctcgagcctc
     2281 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt
     2341 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2401 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     2461 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgcggcg
     2521 ccaatgcatt gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     2581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2641 acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3001 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3061 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3181 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3421 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4741 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4801 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4861 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4921 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4981 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5041 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5101 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5161 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     5221 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agtcgaccgc
     5281 caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct ggacctgtgc
     5341 caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct ggaccccagc
     5401 caattcaata tggcggactt ggcaccatgc caattcaata tggcggacct ggcactgtgc
     5461 caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct tggccctgtg
     5521 ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt
//
