LOCUS       sequence-436923-        7144 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Fluorescent reporter for expressing the KRAS G12D mutant CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7144)
  TITLE     Principles of CRISPR-Cas13 mismatch intolerance enable selective 
            silencing of point-mutated oncogenic RNA with single-base precision
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7144)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: MSCV-KRAS-G12D-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..48
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     176..195
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             395..911
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    975..1316
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1383..1799
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1729..1751
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1767..1783
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             join(1821..1853,1854..1856,1857..2384)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             1821..2384
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             2394..2474
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     misc_feature    2509..3058
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2653..2670)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2880..2899
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(3206..3224)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3474..3493
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             3933..4447
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4609..4625
                     /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(4633..4639,4640..4657,4658..4663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4678..4699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4816..4833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4987..5575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5067..5086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5746..6537,6538..6606))
                     /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     6369..6388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6607..6711)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6779..6797
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6835..6857)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6957..6976
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga
       61 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
      121 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
      181 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
      241 gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc
      301 tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
      361 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
      421 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aatacataac tgagaataga
      481 gaagttcaga tcaaggttag gaacagagag acagcagaat atgggccaaa caggatatct
      541 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc
      601 cgccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa
      661 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct
      721 tctgctcccc gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcctccg
      781 atagactgcg tcgcccgggt acccgtattc ccaataaagc ctcttgctgt ttgcatccga
      841 atcgtggact cgctgatcct tgggagggtc tcctcagatt gattgactgc ccacctcggg
      901 ggtctttcat ttggaggttc caccgagatt tggagacccc tgcccaggga ccaccgaccc
      961 ccccgccggg aggtaagctg gccagcggtc gtttcgtgtc tgtctctgtc tttgtgcgtg
     1021 tttgtgccgg catctaatgt ttgcgcctgc gtctgtacta gttagctaac tagctctgta
     1081 tctggcggac ccgtggtgga actgacgagt tctgaacacc cggccgcaac cctgggagac
     1141 gtcccaggga ctttgggggc cgtttttgtg gcccgacctg aggaagggag tcgatgtgga
     1201 atccgacccc gtcaggatat gtggttctgg taggagacga gaacctaaaa cagttcccgc
     1261 ctccgtctga atttttgctt tcggtttgga accgaagccg cgcgtcttgt ctgctgcagc
     1321 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt
     1381 agggccagac tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc
     1441 ggatcgctca caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg
     1501 cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca
     1561 tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc
     1621 cctacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg
     1681 tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc
     1741 ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccg
     1801 gaattcgttt aaacggatcc atgactgaat ataaacttgt ggtagttgga gctgatggcg
     1861 taggcaagag tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc
     1921 caacaataga ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg
     1981 atattctcga cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga
     2041 ctggggaggg ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc
     2101 accattatag agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag
     2161 taggaaataa atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag
     2221 caagaagtta tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg
     2281 atgccttcta tacattagtt cgagaaattc gaaaacataa agaaaagatg agcaaagatg
     2341 gtaaaaagaa gaaaaagaag tcaaagacaa agtgtgtaat tatgggtagt tcctacccat
     2401 acgatgttcc agattacgct tatccctacg acgtgcctga ttatgcatac ccatatgatg
     2461 tccccgacta tgcctaactc gagcgggatc aattccgccc cccccctaac gttactggcc
     2521 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     2581 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     2641 ggggcctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     2701 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     2761 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     2821 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     2881 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     2941 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     3001 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatacc
     3061 atggtgagca tggacgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     3121 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3181 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     3241 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     3301 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     3361 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     3421 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     3481 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     3541 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     3601 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     3661 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     3721 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta aagcggccgc
     3781 gactctagag tcgacctgca ggcatgcaag cttcaggtag ccggctaacg ttaacaaccg
     3841 gtacctctag aactatagct agcatgcgca aatttaaagc gctgatatcg ataaaataaa
     3901 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     3961 agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa
     4021 gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg
     4081 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc
     4141 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     4201 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     4261 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata
     4321 gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     4381 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     4441 ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat
     4501 attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga
     4561 aatagttcat tatggacagc gcagaaagag ctggggagaa ttgtgaaatt gttatccgct
     4621 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4681 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4741 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4801 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4861 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4921 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4981 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5041 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5101 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5161 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5221 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5281 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5341 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5401 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5461 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5521 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5581 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5641 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5701 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5761 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5821 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5881 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5941 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6001 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6061 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6121 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6181 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6241 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6301 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6361 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6421 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6481 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6541 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6601 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6661 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6721 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6781 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     6841 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     6901 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     6961 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     7021 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     7081 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     7141 gcga
//
