LOCUS       sequence-366431-        5733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KRAS active mutant (G12D 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-G12D
FEATURES             Location/Qualifiers
     source          1..5733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..459
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        460..663
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     614..634
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    665..683
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    686..704
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     714..738
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1411..1440
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1496..1513)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1502..1726
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1898..1917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2049..2096
                     /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             2104..3126
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2104..3126
                     /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    3256..3377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3293..3312)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3347..3366
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3426..3442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3426..3442)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3439..3461)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3450..3466
                     /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(3474..3480,3481..3498,3499..3504))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3519..3540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3657..3674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3828..4416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3908..3927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4587..5378,5379..5447))
                     /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     5210..5229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5448..5552)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5627..5646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgta
       61 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      121 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      181 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      241 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      301 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      361 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      421 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      481 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      541 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      601 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      661 gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt
      721 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      781 caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttcg ccaccatgac
      841 agagtacaaa ctggtggtgg tcggagccga cggcgtgggg aaatccgccc ttaccatcca
      901 gctgatccag aatcacttcg ttgacgaata tgaccccact atagaggact cttaccgaaa
      961 gcaggttgtc atcgacggcg aaacatgcct tttggacatt ctcgacaccg cggggcaaga
     1021 ggagtattct gcaatgaggg accaatacat gcgcactgga gaaggttttc tgtgcgtgtt
     1081 cgctattaat aacaccaagt cgtttgagga tatacaccat tatagggaac agattaaacg
     1141 cgtgaaggat agcgaagatg tacccatggt gctggtaggt aacaaatgtg atttacctag
     1201 cagaacagtc gacactaagc aggcccaaga tctagctagg tcctacggaa tcccattcat
     1261 cgagaccagt gcaaagacac gtcagagagt cgaggatgcc ttttataccc tcgttagaga
     1321 gatccggcag taccggctga aaaagatttc aaaggaagag aagacgccgg ggtgcgtgaa
     1381 aataaagaaa tgtattatca tgtagggccc gaacaaaaac tcatctcaga agaggatctg
     1441 aatagcgccg tcgaccatca tcataatcat cattgagttt aaacccgctg atcagcctcg
     1501 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1561 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1621 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1681 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggagat ctatcgatgt
     1741 tctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     1801 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1861 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1921 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1981 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2041 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     2101 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     2161 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     2221 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     2281 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     2341 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     2401 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     2461 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     2521 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     2581 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     2641 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     2701 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     2761 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     2821 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     2881 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     2941 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     3001 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     3061 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     3121 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3181 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3241 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3301 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3361 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3421 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3481 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3541 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3661 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3721 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3781 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3841 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3901 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3961 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4021 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4081 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4141 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4201 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4261 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4321 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4381 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4441 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4501 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4561 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4621 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4681 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4741 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4801 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4861 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4921 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4981 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5041 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5101 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5161 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5221 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5281 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5341 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5401 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5461 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5521 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5581 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     5641 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     5701 gcgcgagcaa aatttaagct acaacaaggc aag
//
