LOCUS       sequence-409572-        6023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express mouse Kras encoded by native mouse codons and with a G12D
            mutation with an N-terminal FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6023)
  AUTHORS   Le Roux O, Pershing NLK, Kaltenbrun E, Newman NJ, Everitt JI, 
            Baldelli E, Pierobon M, Petricoin EF, Counter CM
  TITLE     Genetically manipulating endogenous Kras levels and oncogenic 
            mutations in vivo influences tissue patterning of murine 
            tumorigenesis.
  JOURNAL   Elife. 2022 Sep 7;11:e75715. doi: 10.7554/eLife.75715.
  PUBMED    36069770
REFERENCE   2  (bases 1 to 6023)
  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: "Elife."; 
            date: "2022-09-7"; volume: "11:e75715. doi"; pages: " 
            10.7554/eLife.75715"
COMMENT     Sequence Label: pcDNA3.1+FLAG-KrasnatG12D
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      944..953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             953..976
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1614..1631)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1620..1844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1890..2318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1977..1996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2187..2208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2327..2347)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2332..2661
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2512..2647
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2574..2593
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2728..3522
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2782..2801)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3392..3411
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3696..3817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3733..3752)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3787..3806
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3866..3882)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3866..3882)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3879..3901)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3890..3906
                     /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(3914..3920,3921..3938,3939..3944))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3959..3980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4097..4114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4268..4856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4348..4367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5027..5818,5819..5887))
                     /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     5650..5669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5888..5992)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccctcgag gccgccacca tggactacaa
      961 agacgatgac gacaagatga ctgagtataa acttgtggtg gttggagctg atggcgtagg
     1021 caagagcgcc ttgacgatac agctaattca gaatcacttt gtggatgagt acgaccctac
     1081 gatagaggac tcctacagga aacaagtagt aattgatgga gaaacctgtc tcttggatat
     1141 tctcgacaca gcaggtcaag aggagtacag tgcaatgagg gaccagtaca tgagaactgg
     1201 ggagggcttt ctttgtgtat ttgccataaa taatactaaa tcatttgaag atattcacca
     1261 ttatagagaa caaattaaaa gagtaaagga ctctgaagat gtgcctatgg tcctggtagg
     1321 gaataagtgt gatttgcctt ctagaacagt agacacgaaa caggctcagg agttagcaag
     1381 gagttacggg attccgttca ttgagacctc agcaaagaca agacagggtg ttgacgatgc
     1441 cttctataca ttagtccgag aaattcgaaa acataaagaa aagatgagca aagatgggaa
     1501 gaagaagaag aagaagtcaa ggacaaggtg tacagttatg tgagaattct gcagatatcc
     1561 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     1621 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1681 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1741 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1801 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1861 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1921 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1981 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2041 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2101 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2161 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2221 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2281 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2341 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2401 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2461 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2521 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2581 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2641 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2701 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2761 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2821 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2881 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2941 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3001 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3061 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3121 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3181 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3241 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3301 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3361 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3421 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3481 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3541 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3601 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3661 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3721 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3781 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3841 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3901 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3961 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4021 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4081 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4141 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4201 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4261 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4321 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4381 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4441 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4501 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4561 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4621 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4681 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4741 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4801 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4861 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4921 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5161 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6001 cccgaaaagt gccacctgac gtc
//
