LOCUS       sequence-436954-        6793 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Fluorescent reporter for expressing a segment of wild type BRAF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6793)
  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  JOURNAL   Unpublished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 6793)
  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            "Unpublished"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-BRAF-WT-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    380..721
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             788..1204
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1134..1156
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1172..1188
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1454..1519
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1563..2112
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1707..1724)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1934..1953
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(2260..2278)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2528..2547
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2987..3501
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3663..3679
                     /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(3687..3693,3694..3711,3712..3717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3732..3753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3870..3887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4041..4629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4121..4140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4800..5591,5592..5660))
                     /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     5423..5442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5661..5765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5833..5851
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5889..5911)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6011..6030
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6224..6246
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6374..6393
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6593..6793,1..316)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
       61 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
      121 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
      181 ctccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt gctgtttgca
      241 tccgaatcgt ggactcgctg atccttggga gggtctcctc agattgattg actgcccacc
      301 tcgggggtct ttcatttgga ggttccaccg agatttggag acccctgccc agggaccacc
      361 gacccccccg ccgggaggta agctggccag cggtcgtttc gtgtctgtct ctgtctttgt
      421 gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg tactagttag ctaactagct
      481 ctgtatctgg cggacccgtg gtggaactga cgagttctga acacccggcc gcaaccctgg
      541 gagacgtccc agggactttg ggggccgttt ttgtggcccg acctgaggaa gggagtcgat
      601 gtggaatccg accccgtcag gatatgtggt tctggtagga gacgagaacc taaaacagtt
      661 cccgcctccg tctgaatttt tgctttcggt ttggaaccga agccgcgcgt cttgtctgct
      721 gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt atttgtctga
      781 aaattagggc cagactgtta ccactccctt aagtttgacc ttaggtcact ggaaagatgt
      841 cgagcggatc gctcacaacc agtcggtaga tgtcaagaag agacgttggg ttaccttctg
      901 ctctgcagaa tggccaacct ttaacgtcgg atggccgcga gacggcacct ttaaccgaga
      961 cctcatcacc caggttaaga tcaaggtctt ttcacctggc ccgcatggac acccagacca
     1021 ggtcccctac atcgtgacct gggaagcctt ggcttttgac ccccctccct gggtcaagcc
     1081 ctttgtacac cctaagcctc cgcctcctct tcctccatcc gccccgtctc tcccccttga
     1141 acctcctcgt tcgaccccgc ctcgatcctc cctttatcca gccctcactc cttctctagg
     1201 cgccggaatt cgccacacca tggattactt acacgccaag tcaatcatcc acagagacct
     1261 caagagtaat aatatatttc ttcatgaaga cctcacagta aaaataggtg attttggtct
     1321 agctacagtg aaatctcgat ggagtgggtc ccatcagttt gaacagttgt ctggatccat
     1381 tttgtggatg gcaccagaag tcatcagaat gcaagataaa aatccataca gctttcagtc
     1441 atcaggtagt tccgattata aggatcatga tggtgattat aaggatcatg atattgacta
     1501 caaagacgat gacgacaaga ccggttaaga actcgagcgg gatcaattcc gccccccccc
     1561 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     1621 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     1681 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     1741 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     1801 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     1861 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     1921 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     1981 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     2041 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     2101 aacacgataa taccatggtg agcatggacg aggaggataa catggccatc atcaaggagt
     2161 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     2221 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     2281 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     2341 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     2401 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     2461 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     2521 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     2581 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     2641 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     2701 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     2761 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     2821 agtaaagcgg ccgcgactct agagtcgacc tgcaggcatg caagcttcag gtagccggct
     2881 aacgttaaca accggtacct ctagaactat agctagcatg cgcaaattta aagcgctgat
     2941 atcgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     3001 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa
     3061 ctgagaatag agaagttcag atcaaggtta ggaacagaga gacagcagaa tatgggccaa
     3121 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     3181 agatgcggtc ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     3241 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     3301 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
     3361 ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     3421 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     3481 ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag
     3541 ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac
     3601 tccaatactc ctgaaatagt tcattatgga cagcgcagaa agagctgggg agaattgtga
     3661 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3721 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3841 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3901 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3961 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4021 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4081 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4141 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4201 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4261 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4321 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4381 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4441 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4501 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4561 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4621 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4681 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4741 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4801 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4861 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4921 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4981 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5041 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5101 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5161 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5221 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5281 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5341 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5401 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5461 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5521 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5581 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5641 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5701 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5761 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5821 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     5881 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     5941 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     6001 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     6061 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     6121 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     6181 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     6241 aaaacgacgg cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6301 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6361 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6421 tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt ccaatttgtt aaagacagga
     6481 tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag cctatagagt
     6541 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     6601 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     6661 tacataactg agaatagaga agttcagatc aaggttagga acagagagac agcagaatat
     6721 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     6781 ggtccccaga tgc
//
