LOCUS       sequence-355621-        5733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KRAS active mutant (G12V mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5733)
  AUTHORS   Vlahos AE, Kang J, Aldrete CA, Zhu R, Chong LS, Elowitz MB, Gao XJ
  TITLE     Protease-controlled secretion and display of intercellular signals.
  JOURNAL   Nat Commun. 2022 Feb 17;13(1):912. doi: 10.1038/s41467-022-28623-y.
  PUBMED    35177637
REFERENCE   2  (bases 1 to 5733)
  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: "Nat 
            Commun."; date: "2022-02-17"; volume: "13(1):912. doi"; pages: " 
            10.1038/s41467-022-28623-y"
COMMENT     Sequence Label: CMVTO-KRAS-G12V
FEATURES             Location/Qualifiers
     source          1..5733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     429..448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(667..771)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(846..865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1032..1411
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1412..1615
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1566..1586
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1617..1635
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1638..1656
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1666..1690
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2363..2392
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2448..2465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2454..2678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2850..2869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3001..3048
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3056..4078
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3056..4078
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4208..4329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4245..4264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4299..4318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4378..4394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4378..4394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4391..4413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4402..4418
                     /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(4426..4432,4433..4450,4451..4456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4471..4492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4609..4626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4780..5368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4860..4879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5539..5733,1..597,598..666))
                     /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"
ORIGIN
        1 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
       61 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
      121 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
      181 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
      241 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
      301 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
      361 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
      421 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
      481 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
      541 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
      601 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
      661 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
      721 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
      781 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
      841 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
      901 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
      961 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg tacgggccag
     1021 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     1081 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1141 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1201 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1261 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1321 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1381 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1441 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1501 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1561 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc
     1621 tatcagtgat agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa
     1681 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga
     1741 ccgatccagc ctccggactc tagcgtttaa acttaagctt cgccaccatg acagagtaca
     1801 aactggtggt ggtcggagcc gtgggcgtgg ggaaatccgc ccttaccatc cagctgatcc
     1861 agaatcactt cgttgacgaa tatgacccca ctatagagga ctcttaccga aagcaggttg
     1921 tcatcgacgg cgaaacatgc cttttggaca ttctcgacac cgcggggcaa gaggagtatt
     1981 ctgcaatgag ggaccaatac atgcgcactg gagaaggttt tctgtgcgtg ttcgctatta
     2041 ataacaccaa gtcgtttgag gatatacacc attataggga acagattaaa cgcgtgaagg
     2101 atagcgaaga tgtacccatg gtgctggtag gtaacaaatg tgatttacct agcagaacag
     2161 tcgacactaa gcaggcccaa gatctagcta ggtcctacgg aatcccattc atcgagacca
     2221 gtgcaaagac acgtcagaga gtcgaggatg ccttttatac cctcgttaga gagatccggc
     2281 agtaccggct gaaaaagatt tcaaaggaag agaagacgcc ggggtgcgtg aaaataaaga
     2341 aatgtattat catgtagggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc
     2401 cgtcgaccat catcataatc atcattgagt ttaaacccgc tgatcagcct cgactgtgcc
     2461 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2521 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2581 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2641 caatagcagg catgctgggg atgcggtggg ctctatggag atctatcgat gttctgggga
     2701 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2761 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2821 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2881 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2941 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta
     3001 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa
     3061 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     3121 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     3181 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     3241 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     3301 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     3361 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     3421 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     3481 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     3541 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     3601 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     3661 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     3721 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     3781 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     3841 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     3901 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     3961 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     4021 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca
     4081 cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4141 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4201 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4261 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4321 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4381 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4441 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4501 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5161 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5221 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5581 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5641 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5701 agatttatca gcaataaacc agccagccgg aag
//
