LOCUS       sequence-430068-        5677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To stably express mouse Kras encoded by native mouse codons and with
            a G12D mutation with an N-terminal FLAG tag in cells resistant to 
            Neo..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5677)
  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 5677)
  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: pBabePuro-FLAG-KrasnatG12D
FEATURES             Location/Qualifiers
     source          1..5677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             942..1358
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1288..1310
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1326..1342
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1378..1387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1387..1410
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2007..2027)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2012..2341
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2192..2327
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2351..2950
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2351..2370)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2847..2867
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3171..3640
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3770..3787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3941..4529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4021..4040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4689..5480,5481..5549))
                     /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     5312..5331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5550..5654)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
      901 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
      961 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1021 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1081 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1141 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1201 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1261 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1321 cctcccttta tccagccctc actccttctc taggcgccgg ccggatccct cgaggccgcc
     1381 accatggact acaaagacga tgacgacaag atgactgagt ataaacttgt ggtggttgga
     1441 gctgatggcg taggcaagag cgccttgacg atacagctaa ttcagaatca ctttgtggat
     1501 gagtacgacc ctacgataga ggactcctac aggaaacaag tagtaattga tggagaaacc
     1561 tgtctcttgg atattctcga cacagcaggt caagaggagt acagtgcaat gagggaccag
     1621 tacatgagaa ctggggaggg ctttctttgt gtatttgcca taaataatac taaatcattt
     1681 gaagatattc accattatag agaacaaatt aaaagagtaa aggactctga agatgtgcct
     1741 atggtcctgg tagggaataa gtgtgatttg ccttctagaa cagtagacac gaaacaggct
     1801 caggagttag caaggagtta cgggattccg ttcattgaga cctcagcaaa gacaagacag
     1861 ggtgttgacg atgccttcta tacattagtc cgagaaattc gaaaacataa agaaaagatg
     1921 agcaaagatg ggaagaagaa gaagaagaag tcaaggacaa ggtgtacagt tatgtgagaa
     1981 ttcgccagca cagtggtcga ccctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2041 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2101 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2161 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2221 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2281 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2341 aaagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     2401 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     2461 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     2521 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     2581 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     2641 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     2701 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     2761 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     2821 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     2881 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     2941 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     3001 tgcatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc
     3061 cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac
     3121 ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg
     3181 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3241 aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     3301 gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca
     3361 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa
     3421 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc
     3481 acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta
     3541 tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc
     3601 tcctctgagt gattgactac ccgtcagcgg gggtctttca catgcagcat gtatcaaaat
     3661 taatttggtt ttttttctta agtatttaca ttaaatggcc atagttgcat taatgaatcg
     3721 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3781 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3841 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3901 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3961 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4021 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4081 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4141 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4201 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4261 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4321 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4381 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4441 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4501 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4561 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4621 tcttcaccta gatccttttg cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg
     4681 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4741 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4801 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4861 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4921 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4981 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5041 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5101 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5161 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5221 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5281 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5341 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5401 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5461 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5521 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5581 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5641 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
