LOCUS       sequence-436934-        6802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for expressing a segment of BRAF-V600E CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6802)
  AUTHORS   Shembrey C, Yang R, Casan J, Hu W, Chen H, Singh GJ, Sadras T, 
            Prasad K, Shortt J, Johnstone RW, Trapani JA, Ekert PG, Fareh M
  TITLE     Principles of CRISPR-Cas13 mismatch intolerance enable selective 
            silencing of point-mutated oncogenic RNA with single-base precision.
  JOURNAL   Sci Adv. 2024 Dec 20;10(51):eadl0731. doi: 10.1126/sciadv.adl0731. 
            Epub 2024 Dec 18.
  PUBMED    39693429
REFERENCE   2  (bases 1 to 6802)
  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: "Sci Adv."; 
            date: "2024-12-20"; volume: "10(51):eadl0731. doi"; pages: " 
            10.1126/sciadv.adl0731. Epub 2024 Dec 18"
COMMENT     Sequence Label: MSCV-BRAF-V600E-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(603..622)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     872..890
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    1038..1587
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1197..1216)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1426..1443
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(1625..1705)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(1955..2371)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(1971..1987)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2003..2025)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    2438..2779
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             2843..3359
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3559..3578)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3706..3728)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3922..3941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4041..4063
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4101..4119)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4187..4291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4292..4360,4361..5152)
                     /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     complement(4510..4529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5323..5911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5812..5831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6065..6082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6199..6220
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6235..6240,6241..6258,6259..6265)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6273..6289
                     /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)."
     LTR             join(6451..6802,1..163)
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 gcaggaactg cttaccacag atatcctgtt tggcccatat tctgctgtct ctctgttcct
       61 aaccttgatc tgaacttctc tattctcagt tatgtatttt ccatgccttg caaaatggcg
      121 ttacttaagc tagcttgcca aacctacagg tggggtcttt cattcccccc tttttctgga
      181 gactaaataa aatcttttat tttatcgata tcagcgcttt aaatttgcgc atgctagcta
      241 tagttctaga ggtaccggtt gttaacgtta gccggctacc tgaagcttgc atgcctgcag
      301 gtcgactcta gagtcgcggc cgctttactt gtacagctcg tccatgccgc cggtggagtg
      361 gcggccctcg gcgcgttcgt actgttccac gatggtgtag tcctcgttgt gggaggtgat
      421 gtccaacttg atgttgacgt tgtaggcgcc gggcagctgc acgggcttct tggccttgta
      481 ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc agcttcagcc tctgcttgat
      541 ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct cccagcccat
      601 ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt
      661 gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca cgccgccgtc
      721 ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct tcaagtagtc
      781 ggggatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact gaggggacag
      841 gatgtcccag gcgaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt
      901 gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga
      961 gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt tatcctcctc
     1021 gtccatgctc accatggtat tatcgtgttt ttcaaaggaa aaccacgtcc ccgtggttcg
     1081 gggggcctag acgttttttt aacctcgact aaacacatgt aaagcatgtg caccgaggcc
     1141 ccagatcaga tcccatacaa tggggtacct tctgggcatc cttcagcccc ttgttgaata
     1201 cgcttgagga gagccatttg actctttcca caactatcca actcacaacg tggcactggg
     1261 gttgtgccgc ctttgcaggt gtatcttata cacgtggctt ttggccgcag aggcacctgt
     1321 cgccaggtgg ggggttccgc tgcctgcaaa gggtcgctac agacgttgtt tgtcttcaag
     1381 aagcttccag aggaactgct tccttcacga cattcaacag accttgcatt cctttggcga
     1441 gaggggaaag acccctagga atgctcgtca agaagacagg gccaggtttc cgggccctca
     1501 cattgccaaa agacggcaat atggtggaaa ataacatata gacaaacgca caccggcctt
     1561 attccaagcg gcttcggcca gtaacgttag ggggggggcg gaattgatcc cgctcgagtt
     1621 cttaggcata gtcggggaca tcatatgggt atgcataatc aggcacgtcg tagggataag
     1681 cgtaatctgg aacatcgtat gggtaggaac tacctgatga ctgaaagctg tatggatttt
     1741 tatcttgcat tctgatgact tctggtgcca tccacaaaat ggatccagac aactgttcaa
     1801 actgatggga cccactccat cgagatttct ctgtagctag accaaaatca cctattttta
     1861 ctgtgaggtc ttcatgaaga aatatattat tactcttgag gtctctgtgg atgattgact
     1921 tggcgtgtaa gtaatccatg gtgtggcgaa ttccggcgcc tagagaagga gtgagggctg
     1981 gataaaggga ggatcgaggc ggggtcgaac gaggaggttc aagggggaga gacggggcgg
     2041 atggaggaag aggaggcgga ggcttagggt gtacaaaggg cttgacccag ggaggggggt
     2101 caaaagccaa ggcttcccag gtcacgatgt aggggacctg gtctgggtgt ccatgcgggc
     2161 caggtgaaaa gaccttgatc ttaacctggg tgatgaggtc tcggttaaag gtgccgtctc
     2221 gcggccatcc gacgttaaag gttggccatt ctgcagagca gaaggtaacc caacgtctct
     2281 tcttgacatc taccgactgg ttgtgagcga tccgctcgac atctttccag tgacctaagg
     2341 tcaaacttaa gggagtggta acagtctggc cctaattttc agacaaatac agaaacacag
     2401 tcagacagag acaacacaga acgatgctgc agcgctgcag cagacaagac gcgcggcttc
     2461 ggttccaaac cgaaagcaaa aattcagacg gaggcgggaa ctgttttagg ttctcgtctc
     2521 ctaccagaac cacatatcct gacggggtcg gattccacat cgactccctt cctcaggtcg
     2581 ggccacaaaa acggccccca aagtccctgg gacgtctccc agggttgcgg ccgggtgttc
     2641 agaactcgtc agttccacca cgggtccgcc agatacagag ctagttagct aactagtaca
     2701 gacgcaggcg caaacattag atgccggcac aaacacgcac aaagacagag acagacacga
     2761 aacgaccgct ggccagctta cctcccggcg ggggggtcgg tggtccctgg gcaggggtct
     2821 ccaaatctcg gtggaacctc caaatgaaag acccccgagg tgggcagtca atcaatctga
     2881 ggagaccctc ccaaggatca gcgagtccac gattcggatg caaacagcaa gaggctttat
     2941 tgggaatacg ggtacccggg cgacgcagtc tatcggagga ctggcgcgcc gagtgagggg
     3001 ttgtgggctc ttttattgag ctcggggagc agaagcgcgc gaacagaagc gagaagcgaa
     3061 ctgattggtt agttcaaata aggcacaggg tcatttcagg tccttggggc accctggaaa
     3121 catctgatgg ttctctagaa actgctgagg gcgggaccgc atctggggac catctgttct
     3181 tggccctgag ccggggcagg aactgcttac cacagatatc ctgtttggcc catattctgc
     3241 tgtctctctg ttcctaacct tgatctgaac ttctctattc tcagttatgt attttccatg
     3301 ccttgcaaaa tggcgttact taagctagct tgccaaacct acaggtgggg tctttcattc
     3361 cccccttttt ctggagacta aataaaatct tttattttat ctatggctcg tactctatag
     3421 gcttcagctg gtgatattgt tgagtcaaaa ctagagcctg gaccactgat atcctgtctt
     3481 taacaaattg gactaatcgc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     3541 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     3601 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     3661 gactgttggg cgccatctcc ttgcatgcac cattccttgc gccgtcgttt tacaacgtcg
     3721 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3781 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3841 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3901 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3961 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4021 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4081 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4141 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4201 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4261 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4321 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4381 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4441 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4501 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4561 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4621 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4681 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4741 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4801 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4861 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4921 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4981 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5041 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5101 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5161 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5221 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5281 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5341 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5401 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5461 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5521 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5581 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5641 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5701 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5761 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5821 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5881 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5941 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6001 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6061 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     6121 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6181 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6241 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacaattc
     6301 tccccagctc tttctgcgct gtccataatg aactatttca ggagtattgg agtatgtgga
     6361 ttcaaaacag tggctaattg agtcaggatt acttaatatt cgactcctac cctcagaatg
     6421 ttgttctcca acttcaagaa actgttaccc atgaaagacc cccgctgacg ggtagtcaat
     6481 cactcagagg agaccctccc aaggaacagc gagaccacaa gtcggatgca actgcaagag
     6541 ggtttattgg atacacgggt acccgggcga cgcagtctat cggaggactg gcgcgccgag
     6601 tgaggggttg tgggctcttt tattgagctc ggggagcaga agcgcgcgaa cagaagcgag
     6661 aagcgaactg attggttagt tcaaataagg cacagggtca tttcaggtcc ttggggcacc
     6721 ctggaaacat ctgatggttc tctagaaact gctgagggcg ggaccgcatc tggggaccat
     6781 ctgttcttgg ccctgagccg gg
//
